CA2385187A1 - Method for observing human endometrial cells in living animals - Google Patents
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Abstract
The invention relates to cells, to an animal model and to methods for observing human endometrial cells in a living animal and more particularly for visualizing human endometriotic lesion implantation, progression and regression in live intact animals through whole body imaging. The invention is based on the non-invasive detection of fluorescent endometrial cells and tissue in dynamic and real-time settings. Animals containing fluorescent endometriotic lesions represent a useful model for studying the mechanisms of the disease, as well as potent tools for pre-clinical in vivo testing of pharmaceutical agents or gene products targeting endometriosis.
Description
METHOD FOR OBSERVING HUMAN ENDOMETRIAL CELLS
IN LIVING ANIMALS
BACKGROUND OF THE INVENTION
(a) Field of the Invention The invention relates to a method for observing human endometrial cells or tissue in a living animal and more particularly for visualizing human endometriotic lesion implantation, progression and regression in live animals. The invention also concerns genetically modified human endometrial cells or tissue, animal models and methods for the study of endometriosis and for evaluating candidate drugs or gene products potentially useful for the treatment of endometriosis.
(b) Description of Prior Art Endometriosis is a gynecological disease characterized by the growth of endometrial tissue at extra-uterine sites. Although benign, it is an aggressive and invalidating disease, linked to several symptoms such as intense, and sometimes chronic, pelvic pain, infertility, deregulated and/or abundant menstrual bleeding (dysmenorrhea), painful intercourse (dyspareunia), diarrhea and/or constipation, in 10 to 15% of women of reproductive age. It is postulated that endometriosis arises from dissemination of endometrial cells in the peritoneal cavity through retrograde menstruation. The exact reasons why, in some individuals, these endometrial cells then adhere to the peritoneal wall, or other organs found within the abdominal cavity, proliferate, induce intense vascularization, and form endometriotic lesions are not well understood. Nonetheless, immunological, hormonal, genetic and environmental factors are suspected to play a role in establishment and maintenance of the disease.
Direct and well-designed in vivo studies on human endometriosis are hampered by practical and ethical considerations. On the other hand, in vitro studies cannot reproduce the three-dimensional organization of endometrial tissue and do not take into account the peculiarity of the peritoneal environment, the major site of endometriotic lesion implantation. Hence, some investigators have used primate animal models for the study of endometriosis, essentially based on spontaneous or chemically-induced formation of endometriotic lesions in the peritoneum of monkeys. Although clinically relevant, these approaches have been limited by both the extensive length of time required for lesions to occur and their prohibitive costs. Therefore, other models, mainly based on the implantation of autologous endometrium tissue, have been developed and validated in rodents such as rabbits, rats and mice. However, these systems are of limited value for the study of the human disease because of the significant histological and biochemical (hormone and cytokine responsiveness, for instance) differences between human and animal endometria. Therefore, the transplantation of human endornetrium into immunodeficient animals (for instance Severe Combined Immunodefic;iency "SCID" or nude mice) has been undertaken in many laboratories. This system was shown to faithfully reproduce the behavior of endometriotic lesions in humans (Bruner et al., 1997. J Clin Invest 99:2851-2857), is amenable to drug testing, and meets practical considerations such as low cost, reliability, easy handling and ethics. Notwithstanding the above, this human-mouse xenograft model still has some caveats. In fact, endometriotic lesions are small (1-2 mm2), not easily distinguishable from the surrounding tissues and their detection is arduous. More importantly, identification and quantification of endometriotic lesions require killing of the animal for each experimental point. This renders this type of model inconvenient for dynamic studies on lesion development or regression after pharmacological treatment in a given animal and multiplies the number of animals to be used. Development of new means to observe human endometriotic lesions in live animals is thus most needed, as they would make of the human-mouse xenograft model the approach of choice for the study of endometriosis and potential treatments.
Until recently, non-invasive imaging techniques have been mainly developed for the visualization of tumor cells in animals. These models are based on complex and expensive instrumentation, not particularly suited for small animals, and often requiring injection of chemical probes to the animals. For instance, tumor cells expressing the luciferase gene can be non-invasively detected in living mice after intraperitoneal injection of luciferine to animals, photon capture by an intensified charge coupled device camera and image processing (see U.S. Patent 5,650,135). Positron emission tomography has also been used for the same purposes, but this system requires that a positron-emitting isotope be specifically retained inside the tumor cells. Furthermore, the resolution routinely achieved with this method is around 6 mm3, too imprecise for the detection of endometriotic lesions.
In the field of endometriosis, the use of external fluorescent labels for the detection of endometriotic lesions in animals has been attempted but fluorescence is very weak and is limited to very short time periods due to rapid diffusion of the probe out of the cells (Tabibzadeh et al., 1999. Front Biosci 4:C4-C9; Yang et a1.,1996. Am J Ob:~tef Gynecol 174:154-160).
There is therefore a long felt need for a method which allows a precise detection of endometriotic lesions in living animal models.
There is also a need for a non-invasive whole-body imaging method which permits to monitor endometriotic lesion formation in living animals, in a dynamic way.
There is also a need fc~r human endometrial tissue or cells genetically modified so as to express a detectable level of a light-emitting protein since direct fluorescent protein production within endometrial tissue has never been achieved.
There is also a need for an animal model for the study of endometriosis which comprises such genetically modified human endometrial tissue.
There is a need also for a method wherein human endometrial cells, genetically modified so as to express a detectable level of a fluorescent protein, are administered to a living animal so as to permit an easy and sensitive visualization of the human cells.
SUMMARY OF THE INVENTION
The invention relates to cells and tissue, animal model and methods for observing human endometrial tissue in a living animal and more particularly for visualizing human endometriotic lesion implantation, progression and regression in live animals.
According to a first aspect, the invention relates to isolated or purified human endometrial cells, or fragments of endometrial tissue, genetically modified so as to express a detectable level of a bioluminescent protein.
According to another aspect, the invention relates to an animal model for ;i the study of endometriosis, comprising isolated or purified human endometrial cells, or fragments of endometriaf tissue as defined previously. This animal model can be used for a dynamic visualization of endometriotic lesion implantation, development and regression, through whole-body imaging. This animal model is very useful as a tool for drug screening and target validation in pre-clinical studies 1 CI of human endometriosis.
A further aspect of the present invention concerns a method for observing human endometrial cells in a living animal. The method comprises the steps of:
- administering sub-cutaneously or intra-peritoneally to a living animal a plurality of human endometrial cells genetically modified so as to express a detectable 15. level of a fluorescent protein; and - observing per-cutaneously fluorescence emitted by said human cells.
According to a more particular aspect, the invention concerns a method for monitoring human endometrial implantation, progression and regression in a living animal. The method comprises the steps of:
20 a) providing a plurality of human endometrial cells, either isolated or in the form of whole endometrial tissue;
b) introducing into these human endometrial cells a nucleotide sequence capable of expressing a detectable level of a fluorescent protein into these human endometrial cells;
25 c) administering sub-cutaneously or intra-peritoneally a plurality of the cells of step (b) to a living animal;
d) allowing the administered cells to form endometrial lesions) in the abdominal cavity of the living animal;
e) exposing the living animal to a light source capable of stimulating or 30 intensifying fluorescence emission by the fluorescent protein expressed in the human fluorescent cells; and f) observing per-cutaneously fluorescence emitted by the human fluorescent cells.
According to a further aspect, the invention relates to a method for assaying a compound efficacy for the treatment of endometriosis. The method :5 comprises the steps of: 1) administering a compound to an animal model as defined previously; and 2) visualizing biological effects of the administered compound onto the human fluorescent endometrial cells of the animal.
An advantage of the present invention is that it provides for the first time human endometrial cells exprEasing a light-emitting protein, thereby conferring a 1!) long-term and intense fluorescence to the cells. Furthermore, the invention allows for the first time a per-cutaneous observation of human endometrial cells in living animals, an approach which is much less invasive than what is commonly used in the art. The cells, animal model and methods of the invention are therefore very useful for the study of endometriosis and for evaluating candidate drugs or gene 1;i products potentially useful for the treatment of endometriosis.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1 A, 1 B, 1 C, 1 D and 1 E are pictures showing GFP expression in fragments of human endometrial biopsies after adenoviral infection.
2(1 Figures 2A and 2B are graphs showing a flow cytometric analysis of GFP
expression in human endometrial cells after adenoviral infection at different multiplicities of infection (M01).
2:i Figures 3A and 3B are bar graphs showing that treatment of endometrial tissue with proteases before infection improves efficacy of human endometrial cells adenoviral infection.
Figures 4A and 4B are pictures showing in vivo imaging of GFP-expressing 3Ci endometriotic lesions established sub-cutaneously in a living nude mouse.
Figures 5A, 5B and 5C are pictures showing in vivo imaging of an intra-peritoneal GFP-expressing endometriotic lesion in a living nude mouse.
DETAILED DESCRIPTION OF THE INVENTION
.5 Tissues and cells The first aspect of thE: invention concerns isolated or purified human endometrial cells, or fragments of endometrial tissue, genetically modified so as to express a detectable level of a bioluminescent (fluorescent) protein.
The isolated or purified human endometrial cells of the invention includes, 1 t) but is not limited to, primary, transformed or immortalized endometrial cells, whole or fragments of endomEarial biopsies, purified endometrial cell subsets, or any human cell giving rise to endometriotic lesions in vivo. Endometrial tissue obtained from endometrial biopsies at any stage of the menstrual cycle, endometrial cell lines or primacy cultures of endometrial cells, transformed or not, 15 or other cells able to produce endornetriotic-like lesions in animals can be used for the practice of the present invention.
The human endometrial cells of the invention are genetically modified so as to express a detectable level of a fluorescent protein. Such modification may be done by the means of well-known methods such as transfection or viral 2(1 infection of a vector encoding the fluorescent protein, be it either extrachromosomal or integrated into - the genome. The invention also encompasses techniques wherein the incorporation of bioluminescent proteins, or the DNA sequences coding such proteins, directly into the cells by the means of methods such as receptor-mediated internalization, liposomes or micro-injection.
2Ci In a preferred embodiment, the cells are infected with an adenovirus with a nucleotide sequence corresponding to the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria. However, the fluorescent protein could be any detectable light emitting protein suitable for tracking endometrial cells non-invasively in a live vertebrate, including but not limited to near-infrared 3Ci probes, proteins with an enhanced fluorescence emission spectrum (e.g.
enhanced green fluorescence protein or EGFP), and proteins with other emission wave lengths or fluorescent-fusion proteins encompassing features similar to GFP or EGFP.
According to another embodiment, the human endometrial cells further comprise a potentially anti-proliferative exogenous nucleotide sequence, i.e.
a ;i sequence potentially capable of reducing, inhibiting, blocking proliferation of the human endometrial cells when expressed in these cells. Examples of exogenous nucleotide sequences include but are not limited to antisense molecules, complete or partial gene candidates (e.g. tyrosine kinase) and any similar molecules with known or potent anti-proliferative or apoptotic activities. The 1 CI biological activity of the exogenous nucleotide sequence (or of a gene product encoded by the same), and proliferation of the endometrial cells may be monitored using well known methods.
Animal model Another aspect of the invention concerns an animal model that may be 1 ~~ used for the study of endometriosis and which comprises isolated or purified genetically modified human endometrial cells as defined previously.
Such an animal is obtained by administering thereto, into diverse locations such as sub-cutaneous or into intra-peritoneal spaces, a plurality of genetically modified human endometrial cells according to the invention. The transplantation 2G of the fluorescent endometrial cells may be done by any suitable means, preferably by the means of injection or surgery. Preferably, these human endometrial cells form endometrial lesions in the abdominal cavity of the animal and implantation, progression and regression of these endometrial lesions may be monitored by observing, through the animal's skin, the fluorescence emitted 25 by the human fluorescent cells.
Preferably, the living animal of the invention consists of an immuno-compromised animal or a syngeneic animal. More preferably, the living animal consists of a rodent, including but not limited to mice, rats, hamsters, guinea pigs, rabbits, or a non-human primate.
Visualization of fluorescent cells The invention allows a non-invasive detection of fluorescent endometrial cells and endometriotic lesian(s) in live animals by visualizing light emitted by these cells or lesion(s).
Preferably, this is achieved by exposing the living animal with fluorescent human endometrial cells to a light source capable of stimulating or intensifying fluorescence emission by the fluorescent protein. Of course the light source must provide an adequate wave-length for the excitation and subsequent fluorescent emission of the fluorescent protein. Results can be viewed by a naked eye, or by using a more sensitive detector. The results may also be recorded through a charge-coupled device camera or through any other image recording system.
Dynamic and real-time visualization of fluorescent endometriotic tissue growing in animals can be repeated many times, for instance for studying responses after drug treatment (see hereinafter). More preferably, the animal is shaved or hairless in order to facilitate light-emission visualization.
Drug screening The animal model and cells of the invention can be used as a tool for drug screening and target validation in pre-clinical studies of human endometriosis.
Therefore, according to another aspect, the invention provides a method for assaying a compound efficacy for the treatment of endometriosis (e.g. a candidate drug or a gene product potentially useful for the prevention or treatment of endometriosis). The method comprises the steps of: 1 ) administering a compound for which efficacy is to be assayed to an animal model as defined previously, and 2) visualizing biological effects of the administered compound onto the fluorescent endometrial cells of this animal. For instance, visualization may consist of observing implantation, progression and regression of endometrial lesions in the animal, or may cansist of measuring fluorescence levels emitted by the human fluorescent cells. Of course, a much simpler method would consists of contacting in vitro the cells of the invention with the compounds) to be assayed.
The present invention will be more readily understood by referring to the following example. This example is illustrative of the wide range of applicability of the present invention and is not intended to limit its scope. Modifications and variations can be made therein without departing from the spirit and scope of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred methods and materials are described.
EXAMPLE 1: In vivo visualization of implantation, pros~ression and rearession of GFP-human endometriotic lesions in mice Material and methods Tissues and cells Human endometrial biopsies were cut in small fragments of 1-2 mm3 with a surgical blade and are kept in culture medium [Dulbecco's modified Eagle's medium/Ham's F12 (Mediatech, Herndon, VA) containing 10% fetal calf serum (FCS), 100 IU/ml penicillin, 100 pg/ml streptomycin, 250 ng/ml Amphotericin B
(Mediatech), and possibly 10 nM 17~-estradiol (Sigma-Aldrich, Oakville, ON, Canada) or other cytokines or hormones]. In some cases, endometrial fragments were incubated for 20 min at 37°C in either 0.05% trypsin, 0.53 mM EDTA
(Wisent), 1 mg/ml collagenase I;type 1A, Sigma) or 1 mg/ml collagenase, 1 mg/ml hyaluronidase (Sigma), in order to slightly disrupt the tissue and favor adenoviral infection. Enzymatic digestion was stopped by the addition of 10% fetal calf serum and extensive washing in culture medium. Ten fragments of endometrial tissue were cultured per well of 48-well plates.
Adenoviral infection and GFP expression In a well of a 48-well plate, ten endometrial fragments were incubated in 100 NI culture medium, alone or in the presence various amounts of purified recombinant adenoviral particlE~s containing the GFPq gene under the CMVS
promoter (AdGFP) (Q-BIOgene, Montreal, QC, Canada). Cultures were incubated for 24 h with continuous orbital agitation at 37°C, 5% C02 in a humidified incubator. GFP expression in whole endometrial fragments was verified by fluorescence microscopy (Leica Canada, Willowdale, ON, Canada) or with a blue light (470 nm) illumination system (Lightools Research, Encinitas, CA).
Quantification of the proportion of GFP-expressing cells was done by flow 5 cytometry (see hereafter).
Mice Five to eight-week old ovariectomized female nude mice were purchased from Harlan-Sprague Dawley (Indianapolis, IN) and kept under specific pathogen 10 free conditions. These immuno-compromised mice were chosen because they are tolerant human tissue xenographs and hairless, facilitating light-emission visualization. Other immuno-compromised mice or syngeneic mice in the case of mouse tissue transplantation can also be used. Twenty-four to 96 h before endometrial tissue transplantation, mice were anesthetized with 2% isofurane and sterile 60-d release capsules containing 1.5 mg 17~i-estradiol (Innovative Research of America, Mendelein, IL) were inserted subcutaneously at a site just below the scapula, in order to maintain constant steroid levels during the whole experiment and optimize endometriotic lesion formation. Mice were always manipulated in sterile conditions.
Infection of human endometrial tissue and non-invasive assessment of lesion formation After the 24 h culture step, endometrial fragments were washed extensively in sterile PBS/2% FCS before transplantation to nude mice. Animals were injected either subcutaneously or intraperitoneally with 10 human endometrial fragments in 200 p1 PBS/2% FCS, with a 18-gauge needle. At different time points after injection, live mice were examined under a blue light source (Lightools Research) for non-invasive (whole body imaging) assessment of lesions. Alternatively, two to three weeks after transplantation, mice were sacrificed and their peritoneum and visceral organs were examined, under blue light illumination, for the removal of endometriotic-like lesions. Lesions were immediately placed in ice-cold culture medium for later analysis by flow cytometry. Images were acquired with a Nikon Coolpix 990T"" digital camera (Nikon Canada, Ville St-Laurent, QC, Canada).
Flow c~rtometric analysis !i Human endometrial fragments infected 24h in vifro with AdGFP were digested with 0.05% trypsin, 53 mM EDTA and 1 mg/ml collagenase in order to obtain single cell suspensions. Cells were extensively washed and resuspended in PBS. GFP fluorochrome was excited at 488 nm and fluorescence was detected at 525 nm. Analysis was performed on a Coulter XLT"" flow cytometer with the XL2T"" software (Coulter Electronics, Ville St-Laurent, QC, Canada).
Results Fluorescence of endometrial fragments cultured for 24h with adenoviral particles carrying the GFPg giene 1 ~~ Development of endometriotic lesions in the mouse is better achieved by the transfer of whole human endometrial fragments, rather than isolated cells, into the peritoneal cavity of animals. In order to preserve the three dimensional structure of the tissue while achieving high levels of fluorescence, in a slowly cycling tissue, adenoviral-mediated gene transfer was used. Furthermore, we chose a "bright" GFP variant, which exhibits 100-fold brighter fluorescence than wild-type GFP and placed it under the control of a strong modified cytomegalovirus promoterlenhancer (CMVS).
A first experiment was carried out to verify the fluorescence of endometrial fragments after infection with GFPq-encoding adenoviral particles. Endometrial fragments were incubated for 24h with different doses of adenoviral particles containing the GFP gene (AdGFP). Typical aspects of GFP-expressing endometrial fragments and cells are shown in Figure 1. Under a fluorescent microscope, intensity and homogeneity of GFP expression at the surface of endometrial biopsies appears to vary between samples, even when identical preparation and dose of AdGFP particles were used {compare Figures 1A, 1 B
and 1 C). On the other hand, cells having detached from endometrial fragments and adhering to the bottom of the well appear more homogeneously infected (Figure 1 D). Also, a macroscopic view of GFP-expressing endometrial fragments, under blue-light illumination is shown in Figure 1 E.
Quantification by flow-cytometry of GFP expression in endometrial cells after adenoviral infection at different multiplicities of infection (M01) To evaluate the proportion of GFP-expressing cells in whole endometrial fragments, we quantified GFP fluorescence by flow cytometry, which analyzes fluorescence at the single cell level. Figure 2A shows the widespread GFP
expression in cells obtained from a pool of 5 endometrial fragments incubated with 500x106 AdGFP particles. In this case, 26% of the cells were GFP-positive, with expression levels extending over three logs of fluorescence. The percentage of GFP-positive cells varied from one experiment to another, probably due to the structure of the target tissue, ranging from 10 to 30% for a viral load of 500x106 particles/well for instance (Figure 2B).
Improvement of adenoviral transduction efficiencv by slight aroteases treatment of endometria) tissue prior to infection fn order to obtain more efficient and homogeneous adenoviral infection, we submitted endometrial fragments to mild enzymatic treatment, thus slightly loosening the tissue and favoring contacts between viral particles and target cells.
Fragments were treated by a mixture of either trypsin/EDTA/collagenase (TC) or hyaluronidase/collagenase (HC) for 20 min at 37°C, before adenoviral infection.
Such treatments did improve infection efficiencies (Figure 3A), and increased the number of GFP-expressing cellls from 15%, without treatment, to 35% and even 50% after HC or TC treatment, respectively. Interestingly, not only more cells expressed GFP after enzymatic treatment, but also higher levels of GFP
fluorescence were achieved (Figure 3B). We later confirmed that enzymatic treatment was not deleterious to the further implantation of endometrial tissue in the mouse peritoneal cavity. -This pre-treatment of endometrial tissue should represent a valuable approach for increasing both the proportion of cells expressing GFP and the global fluorescence of infected fragments, allowing an easier visualization of endometriotic lesions in living animals.
In vivo imaging of GFP-expressing endometriotic lesions established sub-cutaneously in a living nude mouse We determined whetheir GFP levels achieved after adenoviral infection would be sufficient to observe endometriotic lesion formation through the skin.
Mice were injected subcutaneously (s.c.) with ten endometrial fragments infected with 1000x106 viral particles for 24h. It has been shown previously that endometrial fragments injected s.c. in the nude mouse can grow and develop as endometriotic implants on the outer face of the peritoneal membrane, and form little blebs under the skin of the animal. Four days after s.c. injection, a little bulge was readily detectable through the skin of the animal, and appeared fluorescent when observed under a blue light source and through an orange filter, providing adequate excitation and emission filters for GFPq (Figure 4A). After removal of the skin, fluorescence was even more intense (Figure 4B). Of note, although adenoviral DNA does not integrate into the hast genome, expression of the foreign GFP gene remained elevated for a sufficient period of time {at least 3 weeks), allowing establishment and subsequent visualization of endometriotic lesions. This experiment shows that endometrial fragments transduced with the GFP gene by adenoviral infection can survive and implant in vivo, while remaining sufficiently fluorescE~nt in order to be seen through the skin of the animal.
In vivo imaging of an intra-peritoneal GFP-expressing endometriotic lesion in a living nude mouse To assess whether GFP-expressing endometrial tissue injected intraperitoneally could also form lesions visible through the skin of the animal, ten fragments of endometrium cultured with 1000x106 viral particles for 24h were injected. Mice were observed 12 days later. Without exposure to the blue light, no external sign of lesion formation was observed. However, when the mouse was illuminated, a small but distinctive lesion was visible in the pelvis (Figure 5A).
Fluorescence became more evident as the skin (Fig. 5B) and the peritoneum (Fig. 5C) were removed.
While several embodiments of the invention have been described, it will be understood that the present invention is capable of further modifications, and this application is intended to cover any variations, uses, or adaptations of the invention, following in general the principles of the invention and including such departures from the present disclosure as to come within knowledge or customary practice in the art to which the invention pertains, and as may be applied to the essential features hereinbefore set forth and falling within the scope of the invention or the limits of the appended claims.
IN LIVING ANIMALS
BACKGROUND OF THE INVENTION
(a) Field of the Invention The invention relates to a method for observing human endometrial cells or tissue in a living animal and more particularly for visualizing human endometriotic lesion implantation, progression and regression in live animals. The invention also concerns genetically modified human endometrial cells or tissue, animal models and methods for the study of endometriosis and for evaluating candidate drugs or gene products potentially useful for the treatment of endometriosis.
(b) Description of Prior Art Endometriosis is a gynecological disease characterized by the growth of endometrial tissue at extra-uterine sites. Although benign, it is an aggressive and invalidating disease, linked to several symptoms such as intense, and sometimes chronic, pelvic pain, infertility, deregulated and/or abundant menstrual bleeding (dysmenorrhea), painful intercourse (dyspareunia), diarrhea and/or constipation, in 10 to 15% of women of reproductive age. It is postulated that endometriosis arises from dissemination of endometrial cells in the peritoneal cavity through retrograde menstruation. The exact reasons why, in some individuals, these endometrial cells then adhere to the peritoneal wall, or other organs found within the abdominal cavity, proliferate, induce intense vascularization, and form endometriotic lesions are not well understood. Nonetheless, immunological, hormonal, genetic and environmental factors are suspected to play a role in establishment and maintenance of the disease.
Direct and well-designed in vivo studies on human endometriosis are hampered by practical and ethical considerations. On the other hand, in vitro studies cannot reproduce the three-dimensional organization of endometrial tissue and do not take into account the peculiarity of the peritoneal environment, the major site of endometriotic lesion implantation. Hence, some investigators have used primate animal models for the study of endometriosis, essentially based on spontaneous or chemically-induced formation of endometriotic lesions in the peritoneum of monkeys. Although clinically relevant, these approaches have been limited by both the extensive length of time required for lesions to occur and their prohibitive costs. Therefore, other models, mainly based on the implantation of autologous endometrium tissue, have been developed and validated in rodents such as rabbits, rats and mice. However, these systems are of limited value for the study of the human disease because of the significant histological and biochemical (hormone and cytokine responsiveness, for instance) differences between human and animal endometria. Therefore, the transplantation of human endornetrium into immunodeficient animals (for instance Severe Combined Immunodefic;iency "SCID" or nude mice) has been undertaken in many laboratories. This system was shown to faithfully reproduce the behavior of endometriotic lesions in humans (Bruner et al., 1997. J Clin Invest 99:2851-2857), is amenable to drug testing, and meets practical considerations such as low cost, reliability, easy handling and ethics. Notwithstanding the above, this human-mouse xenograft model still has some caveats. In fact, endometriotic lesions are small (1-2 mm2), not easily distinguishable from the surrounding tissues and their detection is arduous. More importantly, identification and quantification of endometriotic lesions require killing of the animal for each experimental point. This renders this type of model inconvenient for dynamic studies on lesion development or regression after pharmacological treatment in a given animal and multiplies the number of animals to be used. Development of new means to observe human endometriotic lesions in live animals is thus most needed, as they would make of the human-mouse xenograft model the approach of choice for the study of endometriosis and potential treatments.
Until recently, non-invasive imaging techniques have been mainly developed for the visualization of tumor cells in animals. These models are based on complex and expensive instrumentation, not particularly suited for small animals, and often requiring injection of chemical probes to the animals. For instance, tumor cells expressing the luciferase gene can be non-invasively detected in living mice after intraperitoneal injection of luciferine to animals, photon capture by an intensified charge coupled device camera and image processing (see U.S. Patent 5,650,135). Positron emission tomography has also been used for the same purposes, but this system requires that a positron-emitting isotope be specifically retained inside the tumor cells. Furthermore, the resolution routinely achieved with this method is around 6 mm3, too imprecise for the detection of endometriotic lesions.
In the field of endometriosis, the use of external fluorescent labels for the detection of endometriotic lesions in animals has been attempted but fluorescence is very weak and is limited to very short time periods due to rapid diffusion of the probe out of the cells (Tabibzadeh et al., 1999. Front Biosci 4:C4-C9; Yang et a1.,1996. Am J Ob:~tef Gynecol 174:154-160).
There is therefore a long felt need for a method which allows a precise detection of endometriotic lesions in living animal models.
There is also a need for a non-invasive whole-body imaging method which permits to monitor endometriotic lesion formation in living animals, in a dynamic way.
There is also a need fc~r human endometrial tissue or cells genetically modified so as to express a detectable level of a light-emitting protein since direct fluorescent protein production within endometrial tissue has never been achieved.
There is also a need for an animal model for the study of endometriosis which comprises such genetically modified human endometrial tissue.
There is a need also for a method wherein human endometrial cells, genetically modified so as to express a detectable level of a fluorescent protein, are administered to a living animal so as to permit an easy and sensitive visualization of the human cells.
SUMMARY OF THE INVENTION
The invention relates to cells and tissue, animal model and methods for observing human endometrial tissue in a living animal and more particularly for visualizing human endometriotic lesion implantation, progression and regression in live animals.
According to a first aspect, the invention relates to isolated or purified human endometrial cells, or fragments of endometrial tissue, genetically modified so as to express a detectable level of a bioluminescent protein.
According to another aspect, the invention relates to an animal model for ;i the study of endometriosis, comprising isolated or purified human endometrial cells, or fragments of endometriaf tissue as defined previously. This animal model can be used for a dynamic visualization of endometriotic lesion implantation, development and regression, through whole-body imaging. This animal model is very useful as a tool for drug screening and target validation in pre-clinical studies 1 CI of human endometriosis.
A further aspect of the present invention concerns a method for observing human endometrial cells in a living animal. The method comprises the steps of:
- administering sub-cutaneously or intra-peritoneally to a living animal a plurality of human endometrial cells genetically modified so as to express a detectable 15. level of a fluorescent protein; and - observing per-cutaneously fluorescence emitted by said human cells.
According to a more particular aspect, the invention concerns a method for monitoring human endometrial implantation, progression and regression in a living animal. The method comprises the steps of:
20 a) providing a plurality of human endometrial cells, either isolated or in the form of whole endometrial tissue;
b) introducing into these human endometrial cells a nucleotide sequence capable of expressing a detectable level of a fluorescent protein into these human endometrial cells;
25 c) administering sub-cutaneously or intra-peritoneally a plurality of the cells of step (b) to a living animal;
d) allowing the administered cells to form endometrial lesions) in the abdominal cavity of the living animal;
e) exposing the living animal to a light source capable of stimulating or 30 intensifying fluorescence emission by the fluorescent protein expressed in the human fluorescent cells; and f) observing per-cutaneously fluorescence emitted by the human fluorescent cells.
According to a further aspect, the invention relates to a method for assaying a compound efficacy for the treatment of endometriosis. The method :5 comprises the steps of: 1) administering a compound to an animal model as defined previously; and 2) visualizing biological effects of the administered compound onto the human fluorescent endometrial cells of the animal.
An advantage of the present invention is that it provides for the first time human endometrial cells exprEasing a light-emitting protein, thereby conferring a 1!) long-term and intense fluorescence to the cells. Furthermore, the invention allows for the first time a per-cutaneous observation of human endometrial cells in living animals, an approach which is much less invasive than what is commonly used in the art. The cells, animal model and methods of the invention are therefore very useful for the study of endometriosis and for evaluating candidate drugs or gene 1;i products potentially useful for the treatment of endometriosis.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1 A, 1 B, 1 C, 1 D and 1 E are pictures showing GFP expression in fragments of human endometrial biopsies after adenoviral infection.
2(1 Figures 2A and 2B are graphs showing a flow cytometric analysis of GFP
expression in human endometrial cells after adenoviral infection at different multiplicities of infection (M01).
2:i Figures 3A and 3B are bar graphs showing that treatment of endometrial tissue with proteases before infection improves efficacy of human endometrial cells adenoviral infection.
Figures 4A and 4B are pictures showing in vivo imaging of GFP-expressing 3Ci endometriotic lesions established sub-cutaneously in a living nude mouse.
Figures 5A, 5B and 5C are pictures showing in vivo imaging of an intra-peritoneal GFP-expressing endometriotic lesion in a living nude mouse.
DETAILED DESCRIPTION OF THE INVENTION
.5 Tissues and cells The first aspect of thE: invention concerns isolated or purified human endometrial cells, or fragments of endometrial tissue, genetically modified so as to express a detectable level of a bioluminescent (fluorescent) protein.
The isolated or purified human endometrial cells of the invention includes, 1 t) but is not limited to, primary, transformed or immortalized endometrial cells, whole or fragments of endomEarial biopsies, purified endometrial cell subsets, or any human cell giving rise to endometriotic lesions in vivo. Endometrial tissue obtained from endometrial biopsies at any stage of the menstrual cycle, endometrial cell lines or primacy cultures of endometrial cells, transformed or not, 15 or other cells able to produce endornetriotic-like lesions in animals can be used for the practice of the present invention.
The human endometrial cells of the invention are genetically modified so as to express a detectable level of a fluorescent protein. Such modification may be done by the means of well-known methods such as transfection or viral 2(1 infection of a vector encoding the fluorescent protein, be it either extrachromosomal or integrated into - the genome. The invention also encompasses techniques wherein the incorporation of bioluminescent proteins, or the DNA sequences coding such proteins, directly into the cells by the means of methods such as receptor-mediated internalization, liposomes or micro-injection.
2Ci In a preferred embodiment, the cells are infected with an adenovirus with a nucleotide sequence corresponding to the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria. However, the fluorescent protein could be any detectable light emitting protein suitable for tracking endometrial cells non-invasively in a live vertebrate, including but not limited to near-infrared 3Ci probes, proteins with an enhanced fluorescence emission spectrum (e.g.
enhanced green fluorescence protein or EGFP), and proteins with other emission wave lengths or fluorescent-fusion proteins encompassing features similar to GFP or EGFP.
According to another embodiment, the human endometrial cells further comprise a potentially anti-proliferative exogenous nucleotide sequence, i.e.
a ;i sequence potentially capable of reducing, inhibiting, blocking proliferation of the human endometrial cells when expressed in these cells. Examples of exogenous nucleotide sequences include but are not limited to antisense molecules, complete or partial gene candidates (e.g. tyrosine kinase) and any similar molecules with known or potent anti-proliferative or apoptotic activities. The 1 CI biological activity of the exogenous nucleotide sequence (or of a gene product encoded by the same), and proliferation of the endometrial cells may be monitored using well known methods.
Animal model Another aspect of the invention concerns an animal model that may be 1 ~~ used for the study of endometriosis and which comprises isolated or purified genetically modified human endometrial cells as defined previously.
Such an animal is obtained by administering thereto, into diverse locations such as sub-cutaneous or into intra-peritoneal spaces, a plurality of genetically modified human endometrial cells according to the invention. The transplantation 2G of the fluorescent endometrial cells may be done by any suitable means, preferably by the means of injection or surgery. Preferably, these human endometrial cells form endometrial lesions in the abdominal cavity of the animal and implantation, progression and regression of these endometrial lesions may be monitored by observing, through the animal's skin, the fluorescence emitted 25 by the human fluorescent cells.
Preferably, the living animal of the invention consists of an immuno-compromised animal or a syngeneic animal. More preferably, the living animal consists of a rodent, including but not limited to mice, rats, hamsters, guinea pigs, rabbits, or a non-human primate.
Visualization of fluorescent cells The invention allows a non-invasive detection of fluorescent endometrial cells and endometriotic lesian(s) in live animals by visualizing light emitted by these cells or lesion(s).
Preferably, this is achieved by exposing the living animal with fluorescent human endometrial cells to a light source capable of stimulating or intensifying fluorescence emission by the fluorescent protein. Of course the light source must provide an adequate wave-length for the excitation and subsequent fluorescent emission of the fluorescent protein. Results can be viewed by a naked eye, or by using a more sensitive detector. The results may also be recorded through a charge-coupled device camera or through any other image recording system.
Dynamic and real-time visualization of fluorescent endometriotic tissue growing in animals can be repeated many times, for instance for studying responses after drug treatment (see hereinafter). More preferably, the animal is shaved or hairless in order to facilitate light-emission visualization.
Drug screening The animal model and cells of the invention can be used as a tool for drug screening and target validation in pre-clinical studies of human endometriosis.
Therefore, according to another aspect, the invention provides a method for assaying a compound efficacy for the treatment of endometriosis (e.g. a candidate drug or a gene product potentially useful for the prevention or treatment of endometriosis). The method comprises the steps of: 1 ) administering a compound for which efficacy is to be assayed to an animal model as defined previously, and 2) visualizing biological effects of the administered compound onto the fluorescent endometrial cells of this animal. For instance, visualization may consist of observing implantation, progression and regression of endometrial lesions in the animal, or may cansist of measuring fluorescence levels emitted by the human fluorescent cells. Of course, a much simpler method would consists of contacting in vitro the cells of the invention with the compounds) to be assayed.
The present invention will be more readily understood by referring to the following example. This example is illustrative of the wide range of applicability of the present invention and is not intended to limit its scope. Modifications and variations can be made therein without departing from the spirit and scope of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred methods and materials are described.
EXAMPLE 1: In vivo visualization of implantation, pros~ression and rearession of GFP-human endometriotic lesions in mice Material and methods Tissues and cells Human endometrial biopsies were cut in small fragments of 1-2 mm3 with a surgical blade and are kept in culture medium [Dulbecco's modified Eagle's medium/Ham's F12 (Mediatech, Herndon, VA) containing 10% fetal calf serum (FCS), 100 IU/ml penicillin, 100 pg/ml streptomycin, 250 ng/ml Amphotericin B
(Mediatech), and possibly 10 nM 17~-estradiol (Sigma-Aldrich, Oakville, ON, Canada) or other cytokines or hormones]. In some cases, endometrial fragments were incubated for 20 min at 37°C in either 0.05% trypsin, 0.53 mM EDTA
(Wisent), 1 mg/ml collagenase I;type 1A, Sigma) or 1 mg/ml collagenase, 1 mg/ml hyaluronidase (Sigma), in order to slightly disrupt the tissue and favor adenoviral infection. Enzymatic digestion was stopped by the addition of 10% fetal calf serum and extensive washing in culture medium. Ten fragments of endometrial tissue were cultured per well of 48-well plates.
Adenoviral infection and GFP expression In a well of a 48-well plate, ten endometrial fragments were incubated in 100 NI culture medium, alone or in the presence various amounts of purified recombinant adenoviral particlE~s containing the GFPq gene under the CMVS
promoter (AdGFP) (Q-BIOgene, Montreal, QC, Canada). Cultures were incubated for 24 h with continuous orbital agitation at 37°C, 5% C02 in a humidified incubator. GFP expression in whole endometrial fragments was verified by fluorescence microscopy (Leica Canada, Willowdale, ON, Canada) or with a blue light (470 nm) illumination system (Lightools Research, Encinitas, CA).
Quantification of the proportion of GFP-expressing cells was done by flow 5 cytometry (see hereafter).
Mice Five to eight-week old ovariectomized female nude mice were purchased from Harlan-Sprague Dawley (Indianapolis, IN) and kept under specific pathogen 10 free conditions. These immuno-compromised mice were chosen because they are tolerant human tissue xenographs and hairless, facilitating light-emission visualization. Other immuno-compromised mice or syngeneic mice in the case of mouse tissue transplantation can also be used. Twenty-four to 96 h before endometrial tissue transplantation, mice were anesthetized with 2% isofurane and sterile 60-d release capsules containing 1.5 mg 17~i-estradiol (Innovative Research of America, Mendelein, IL) were inserted subcutaneously at a site just below the scapula, in order to maintain constant steroid levels during the whole experiment and optimize endometriotic lesion formation. Mice were always manipulated in sterile conditions.
Infection of human endometrial tissue and non-invasive assessment of lesion formation After the 24 h culture step, endometrial fragments were washed extensively in sterile PBS/2% FCS before transplantation to nude mice. Animals were injected either subcutaneously or intraperitoneally with 10 human endometrial fragments in 200 p1 PBS/2% FCS, with a 18-gauge needle. At different time points after injection, live mice were examined under a blue light source (Lightools Research) for non-invasive (whole body imaging) assessment of lesions. Alternatively, two to three weeks after transplantation, mice were sacrificed and their peritoneum and visceral organs were examined, under blue light illumination, for the removal of endometriotic-like lesions. Lesions were immediately placed in ice-cold culture medium for later analysis by flow cytometry. Images were acquired with a Nikon Coolpix 990T"" digital camera (Nikon Canada, Ville St-Laurent, QC, Canada).
Flow c~rtometric analysis !i Human endometrial fragments infected 24h in vifro with AdGFP were digested with 0.05% trypsin, 53 mM EDTA and 1 mg/ml collagenase in order to obtain single cell suspensions. Cells were extensively washed and resuspended in PBS. GFP fluorochrome was excited at 488 nm and fluorescence was detected at 525 nm. Analysis was performed on a Coulter XLT"" flow cytometer with the XL2T"" software (Coulter Electronics, Ville St-Laurent, QC, Canada).
Results Fluorescence of endometrial fragments cultured for 24h with adenoviral particles carrying the GFPg giene 1 ~~ Development of endometriotic lesions in the mouse is better achieved by the transfer of whole human endometrial fragments, rather than isolated cells, into the peritoneal cavity of animals. In order to preserve the three dimensional structure of the tissue while achieving high levels of fluorescence, in a slowly cycling tissue, adenoviral-mediated gene transfer was used. Furthermore, we chose a "bright" GFP variant, which exhibits 100-fold brighter fluorescence than wild-type GFP and placed it under the control of a strong modified cytomegalovirus promoterlenhancer (CMVS).
A first experiment was carried out to verify the fluorescence of endometrial fragments after infection with GFPq-encoding adenoviral particles. Endometrial fragments were incubated for 24h with different doses of adenoviral particles containing the GFP gene (AdGFP). Typical aspects of GFP-expressing endometrial fragments and cells are shown in Figure 1. Under a fluorescent microscope, intensity and homogeneity of GFP expression at the surface of endometrial biopsies appears to vary between samples, even when identical preparation and dose of AdGFP particles were used {compare Figures 1A, 1 B
and 1 C). On the other hand, cells having detached from endometrial fragments and adhering to the bottom of the well appear more homogeneously infected (Figure 1 D). Also, a macroscopic view of GFP-expressing endometrial fragments, under blue-light illumination is shown in Figure 1 E.
Quantification by flow-cytometry of GFP expression in endometrial cells after adenoviral infection at different multiplicities of infection (M01) To evaluate the proportion of GFP-expressing cells in whole endometrial fragments, we quantified GFP fluorescence by flow cytometry, which analyzes fluorescence at the single cell level. Figure 2A shows the widespread GFP
expression in cells obtained from a pool of 5 endometrial fragments incubated with 500x106 AdGFP particles. In this case, 26% of the cells were GFP-positive, with expression levels extending over three logs of fluorescence. The percentage of GFP-positive cells varied from one experiment to another, probably due to the structure of the target tissue, ranging from 10 to 30% for a viral load of 500x106 particles/well for instance (Figure 2B).
Improvement of adenoviral transduction efficiencv by slight aroteases treatment of endometria) tissue prior to infection fn order to obtain more efficient and homogeneous adenoviral infection, we submitted endometrial fragments to mild enzymatic treatment, thus slightly loosening the tissue and favoring contacts between viral particles and target cells.
Fragments were treated by a mixture of either trypsin/EDTA/collagenase (TC) or hyaluronidase/collagenase (HC) for 20 min at 37°C, before adenoviral infection.
Such treatments did improve infection efficiencies (Figure 3A), and increased the number of GFP-expressing cellls from 15%, without treatment, to 35% and even 50% after HC or TC treatment, respectively. Interestingly, not only more cells expressed GFP after enzymatic treatment, but also higher levels of GFP
fluorescence were achieved (Figure 3B). We later confirmed that enzymatic treatment was not deleterious to the further implantation of endometrial tissue in the mouse peritoneal cavity. -This pre-treatment of endometrial tissue should represent a valuable approach for increasing both the proportion of cells expressing GFP and the global fluorescence of infected fragments, allowing an easier visualization of endometriotic lesions in living animals.
In vivo imaging of GFP-expressing endometriotic lesions established sub-cutaneously in a living nude mouse We determined whetheir GFP levels achieved after adenoviral infection would be sufficient to observe endometriotic lesion formation through the skin.
Mice were injected subcutaneously (s.c.) with ten endometrial fragments infected with 1000x106 viral particles for 24h. It has been shown previously that endometrial fragments injected s.c. in the nude mouse can grow and develop as endometriotic implants on the outer face of the peritoneal membrane, and form little blebs under the skin of the animal. Four days after s.c. injection, a little bulge was readily detectable through the skin of the animal, and appeared fluorescent when observed under a blue light source and through an orange filter, providing adequate excitation and emission filters for GFPq (Figure 4A). After removal of the skin, fluorescence was even more intense (Figure 4B). Of note, although adenoviral DNA does not integrate into the hast genome, expression of the foreign GFP gene remained elevated for a sufficient period of time {at least 3 weeks), allowing establishment and subsequent visualization of endometriotic lesions. This experiment shows that endometrial fragments transduced with the GFP gene by adenoviral infection can survive and implant in vivo, while remaining sufficiently fluorescE~nt in order to be seen through the skin of the animal.
In vivo imaging of an intra-peritoneal GFP-expressing endometriotic lesion in a living nude mouse To assess whether GFP-expressing endometrial tissue injected intraperitoneally could also form lesions visible through the skin of the animal, ten fragments of endometrium cultured with 1000x106 viral particles for 24h were injected. Mice were observed 12 days later. Without exposure to the blue light, no external sign of lesion formation was observed. However, when the mouse was illuminated, a small but distinctive lesion was visible in the pelvis (Figure 5A).
Fluorescence became more evident as the skin (Fig. 5B) and the peritoneum (Fig. 5C) were removed.
While several embodiments of the invention have been described, it will be understood that the present invention is capable of further modifications, and this application is intended to cover any variations, uses, or adaptations of the invention, following in general the principles of the invention and including such departures from the present disclosure as to come within knowledge or customary practice in the art to which the invention pertains, and as may be applied to the essential features hereinbefore set forth and falling within the scope of the invention or the limits of the appended claims.
Claims (26)
1. A method for observing human endometrial cells in a living animal, comprising the steps of:
- administering sub-cutaneously or intra-peritoneally a plurality of human endometrial cells to said living animal, wherein said human endometrial cells have been genetically modified so as to express a detectable level of a fluorescent protein; and - observing per-cutaneously fluorescence emitted by said human cells.
- administering sub-cutaneously or intra-peritoneally a plurality of human endometrial cells to said living animal, wherein said human endometrial cells have been genetically modified so as to express a detectable level of a fluorescent protein; and - observing per-cutaneously fluorescence emitted by said human cells.
2. The method of claim 1, further comprising the step of exposing said living animal to a light source capable of stimulating or intensifying fluorescence emission by said fluorescent protein.
3. The method of claim 1 or 2, wherein the human endometrial cells administered form at least one endometrial lesion in the abdominal cavity of said living animal, and wherein the observing steps further comprise monitoring implantation, progression and regression of said at least one endometrial lesion.
4. The method of any one of claims 1 to 3, wherein said human endometrial cells are selected from the group consisting of primary isolated human endometrial cells, transformed human endometrial cells and immortalized human endometrial cells.
5. The method of any one of claims 1 to 4, wherein said genetic modification consists of introducing into said cell a nucleotide sequence encoding said fluorescent protein.
6. The method of claim 5, wherein the genetic modification of said human endometrial cells is a genetic modification conferring a long-term and intense fluorescence to said cells.
7. The method of any one of claims 1 to 6, wherein said fluorescent protein is selected from the group consisting of GFP, EGFP, and fusion-proteins thereof.
8. The method of any one of claims 1 to 7, wherein the administration step consists of an injection or a surgical implantation of said human endometrial cells administration.
9. The method of any one of claims 1 to 8, wherein said living animal consists of an immuno-compromised animal or a syngeneic animal.
10. The method of any one of claims 1 to 9, wherein said living animal consists of a rodent or a non human primate.
11. The method of claim 10, wherein said rodent is selected from the group consisting of mice, rat, hamster, guinea pig, and rabbit.
12. The method of any one of claims 7 to 11, further comprising the steps of:
1) administering a compound to said mammal, and 2) monitoring biological effects of said compound on said endometrial human cells.
1) administering a compound to said mammal, and 2) monitoring biological effects of said compound on said endometrial human cells.
13. The method of claim 12, for screening a drug capable of reducing, inhibiting or blocking proliferation of said human endometrial cells.
14. The method of any one of claims 1 to 13, wherein said human endometrial cells further comprise a potentially anti-proliferative exogenous nucleotide sequence.
15. The method of claim 14, further comprising the step of monitoring biological activity of said exogenous nucleotide sequence.
16. The method of claim 14 or 15, wherein said exogenous nucleotide sequence consists of a gene candidate potentially capable of reducing, inhibiting,
17 blocking or increasing proliferation of said human endometrial cells when expressed therein.
17. A method for monitoring human endometrial implantation, progression and regression in a living animal, comprising the steps of:
a) providing a plurality of human endometrial cells;
b) introducing into said human endometrial cells a nucleotide sequence capable of expressing a detectable level of a fluorescent protein into said cells;
c) administering sub-cutaneously or intra-peritoneally a plurality of the cells of step (b) to said living animal;
d) allowing said administered cells to form at least one endometrial lesion in the abdominal cavity of said living animal;
e) exposing said living animal to a light source capable of stimulating or intensifying fluorescence emission by the fluorescent protein expressed in said endometrial human cells; and f) observing per-cutaneously fluorescence emitted by said human cells.
17. A method for monitoring human endometrial implantation, progression and regression in a living animal, comprising the steps of:
a) providing a plurality of human endometrial cells;
b) introducing into said human endometrial cells a nucleotide sequence capable of expressing a detectable level of a fluorescent protein into said cells;
c) administering sub-cutaneously or intra-peritoneally a plurality of the cells of step (b) to said living animal;
d) allowing said administered cells to form at least one endometrial lesion in the abdominal cavity of said living animal;
e) exposing said living animal to a light source capable of stimulating or intensifying fluorescence emission by the fluorescent protein expressed in said endometrial human cells; and f) observing per-cutaneously fluorescence emitted by said human cells.
18. The method of claim 17, wherein said living animal consists of an immuno-compromised or a syngeneic rodent.
19. An isolated or purified human endometrial cell genetically modified so as to express a detectable level of a fluorescent protein.
20. An animal model for the study of endometriosis, comprising human endometrial cells genetically modified so as to express a detectable level of a fluorescent protein.
21. The animal model of claim 20, wherein said human endometrial cells have formed at least one endometrial lesion in the abdominal cavity of said animal.
22. A method for assaying a compound efficacy for the treatment of endometriosis, comprising the steps of: 1) administering said compound to an animal model as defined in claim 20 or 21; and 2) visualizing biological effects of said compound onto the fluorescent endometrial cells of said animal.
23. The method of claim 22, wherein said compound consists of a candidate drug or a gene product potentially useful for the prevention or treatment of endometriosis.
24. The method of claim 22 or 23, wherein the visualization step consists of observing implantation, progression and regression of endometrial lesions in said animal.
25. The method of any one of claims 22 to 24, further comprising the steps of:
1) introducing into said human endometrial cells an exogenous nucleotide sequence with potential anti-proliferative biological activity; and 2) monitoring the biological activity of said exogenous nucleotide sequence.
1) introducing into said human endometrial cells an exogenous nucleotide sequence with potential anti-proliferative biological activity; and 2) monitoring the biological activity of said exogenous nucleotide sequence.
26. The method of claim 25, wherein said exogenous nucleotide sequence consists of a gene candidate potentially capable of reducing, inhibiting, blocking or increasing proliferation of said human endometrial cells when expressed therein.
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| US28880401P | 2001-05-07 | 2001-05-07 | |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110915759A (en) * | 2019-12-17 | 2020-03-27 | 江西省妇幼保健院 | Construction method of endometriosis animal model |
| CN117801091A (en) * | 2023-12-28 | 2024-04-02 | 广东圆康再生医学科技开发有限公司 | Application of endometrial stem cells in endometrial repair |
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2002
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110915759A (en) * | 2019-12-17 | 2020-03-27 | 江西省妇幼保健院 | Construction method of endometriosis animal model |
| CN110915759B (en) * | 2019-12-17 | 2022-12-13 | 江西省妇幼保健院 | Construction method of endometriosis animal model |
| CN117801091A (en) * | 2023-12-28 | 2024-04-02 | 广东圆康再生医学科技开发有限公司 | Application of endometrial stem cells in endometrial repair |
| CN117801091B (en) * | 2023-12-28 | 2024-05-31 | 广东圆康再生医学科技开发有限公司 | Application of endometrial stem cells in endometrial repair |
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