Patent Application
20100034779
The invention relates to the isolation and culture of adult stem cells. More specifically, the invention relates to adult spermatogonial stem cells as well as the culture of embryonic stem cell-like cells (ESCs), embryoid bodies, and differentiated cells or tissues derived from adult spermatogonial stem cells.
In the mouse, the embryonic precursors of the gametes, known as primordial germ cells (PGCs), arising from embryonal ectoderm are identified in the embryo around day 7 post-coitum. Over the next few days, they proliferate during migration and reach the genital ridges. In males, they enter foetal seminiferous tubules, become gonocytes, and cease dividing. Following birth in mammals, gonocytes migrate to the seminiferous tubule basement membrane and develop to type A spermatogonia, the spermatogonial stem cells (SSCs). SSCs have the capability to self-renew and to produce large numbers of progenitors that are destined to differentiate into spermatozoa throughout life (Spradling, A. et al., Nature 414, 2001, 98-104). Embryonic germ cells (EGCs) derived from PGCs are pluripotent and have differentiation potential similar to embryonic stem cells (ESCs) (Resnick, J. L. et al., Nature 359, 1992, 550-1; Matsui, Y. et al., Cell 70, 1992, 841-7.). Germline stem cells from neonatal (0-2 days old) mouse testis are also pluripotent and able to differentiate into various types of somatic cells (Kanatsu-Shinohara, M. et al., Cell 119, 2004, 1001-12) suggesting that the germline lineage may retain the ability to generate pluripotent cells. However, until now there has been no evidence for the pluripotency and plasticity of adult spermatogonial stem cells (SSCs), which are responsible for maintaining spermatogenesis throughout life in the male (Spradling, 2001, supra).
In a previous study by Kanatsu-Shinohara et al. (2004, supra) pluripotent ES-like cells expressing the c-kit antigen were successfully generated from neonatal mouse testis and induced to differentiate further into various tissue types. The authors also attempted to obtain ES-like cells from an enriched preparation of adult spermatogonial stem cells derived from wildtype mouse testis, but no ES-like cells could be obtained under the culture conditions used therein. However, the authors could obtain ES-like cells in two of eight experiments from p53 knockout mice. Since knockout of p53 results in enhanced tumorigenesis (Donehower, L. A., et al., Mol Carcinog 1995, 14, 16-22), and the cells are not characterized by Kanatsu-Shinohara et al. (2004, supra), the origin of the ES-like cells is not clear.
U.S. Pat. No. 6,251,671 (Hogan et al.) suggests a method of making a population of mammalian pluripotent embryonic stem cells by culturing neonatal or postnatal SSCs either in the presence of a culture medium containing BMP8 protein and/or growth enhancing amounts of basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), and membrane associated and soluble steel factors, the latter constituting ligands for the c-kit receptor. However, no further characterization of cells obtained in such a way is shown, and no method of further differentiating such cells into embryoid bodies and/or differentiated cells is mentioned in the document.
Pluripotent ESC-like cells obtained from adult spermatogonial stem cells may have great potential for cell-based organ regeneration therapy due to their origin from adult tissue. Since these cells are pluripotent like ESCs, use of their differentiated derivatives for autologous transplantation may allow establishment of individual cell-based therapy without the ethical problems associated with human ESCs. In addition to cell transplantation strategies, use of ESC-like cells derived from SSCs of transgenic animals or from testicular biopsies of humans with genetic abnormalities or varieties might allow developing various cell types for studying physiological or pathophysiological processes under in vitro conditions.
One object of the present invention therefore is to establish new cell culture conditions to which isolated adult spermatogonial stem cells (SSCs) respond in a reproducible manner and acquire embryonic stem cell (ESC)-like properties such as the ability to differentiate spontaneously into derivatives of three embryonic germ layers in vitro and in vivo. Culture conditions for the initial culture of testicular cells used until now included subjecting the cells to a variety of growth factors which may result in the overgrowth of other cells, including fibroblasts, endothelial and Sertoli cells.
Another object of the present invention is to provide ESC-like cells and embryoid bodies derived from adult spermatogonial stem cells for producing a tissue or a differentiated cell that may be used in cell-based organ-regeneration therapy and autologous or allogenic cell, tissue, or organ transplantation.
Accordingly, the present invention relates to a method of producing embryonic stem cell (ESC)-like cells derived from adult mammalian testis, as defined in the claims. Furthermore, the invention relates to a method of producing embryoid bodies from ESC-like cells as well as a method of producing a tissue and/or a differentiated cell from the ESC-like cell or the embryoid body, as defined in the claims. In addition, an ESC-like cell, an embryoid body and/or differentiated cell and/or tissue obtainable by said methods and pharmaceutical preparations containing the same are provided, as defined in the claims. Finally, the invention relates to the use of these products for medical treatments and the preparation of pharmaceutical compositions for medical treatment, respectively, as specified in the claims.
The present invention accomplishes the establishment of cell culture conditions containing only the growth factors and feeder cells essential for the successful proliferation of adult SSCs, and it was surprisingly found that thus cultured SSCs can develop into ESC-like cells and further differentiated cell and tissue forms.
In a first aspect, the present invention relates to a method of producing embryonic stem cell (ESC)-like cells derived from adult mammalian testis, comprising the steps of (a) propagating isolated mammalian testicular cells (i) in a suitable culture medium containing serum or serum replacement and optionally containing one or more growth factors selected from the group consisting of leukaemia inhibitory factor (LIF), glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), (2′Z, 3′E)-6-Bromoindirubin-3′-oxime (BIO; GSK-3 inhibitor IX) and B27, and/or (ii) in a suitable culture medium containing serum or serum replacement on a suitable feeder layer; for a period sufficient to allow the formation of ESC-like cells; and (b) optionally obtaining the ESC-like cells from the culture.
The “ESC-like cells” obtained according to the above method exhibit properties of embryonic stem cells (ESCs). In particular, they may give rise to cells derived from all three embryonic germ layers, i.e. mesoderm, endoderm, and ectoderm and, thus, all cells of the body. The ESC-like cells are derived from adult mammalian testis. In a first step, isolated mammalian testicular cells are propagated in a suitable culture medium. The testicular cells can be obtained for example from testis biopsies and may be isolated as described in the experimental section herein. Further methods for the isolation of such cells are known to the skilled person (see, e.g., U.S. Pat. No. 6,251,671).
The thus isolated testicular cells are then transferred in a suitable cell culture medium for continued growth and expansion. Typically, the cells are further passaged upon reaching about 40 to 70% confluency. Usually, cells from human, non-human primate and mouse are passaged every 1-7 days. ESC-like cells can be obtained after 2 to 3 passages in case of murine cells, and after 5-7 passages in case of human or non-human primate cells. In principle, it is possible to culture these ESC-like cells up to 30 passages and longer.
By monitoring the appearance of tightly packed ESC-like colonies, as those shown in the appended drawings, the skilled person can readily determine whether a sufficient number of cells have been obtained so that the propagating may be terminated. Methods to retrieve single ESC-like colonies include picking them by micromanipulation from the culture under microscope supervision. It is, in principle, possible to culture these ESC-like cells up to 30 passages and longer.
Typical examples of suitable culture mediums are commercially available DMEM, DMEM/F12 and KnockOut™ D-MEM (GIBCO®) media. For human or non-human primate cells DMEM/F12 medium is generally used, whereas for mouse cells DMEM is typically used. Alternative media are available to the skilled person from various commercial sources and can be tested for their positive effect on the growth of the desired cells. The medium may be supplemented with serum or serum replacement. The serum may be Fetal Calf Serum (FCS), or an equivalent serum derived from other species such as human, horse and pig. Alternatively, a commercially available synthetic serum replacement such as KnockOut™ Serum Replacement (GIBCO®) may be used. The serum or serum replacement may be contained in the respective medium in a quantity of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30%, in particular 5, 10, 15, 20 and 25% (v/v). The medium may also contain additional additives such as glutamine, non essential amino acids, β-mercaptoethanol, and antibiotics, all of which are known to the skilled person and can, thus, be optimized in accordance with standard procedures.
In addition, the medium may contain one or more growth factors, supporting the growth of the desired ESC-like cells. Typical examples are leukaemia inhibitory factor (LIF), glial cell line-derived neurotrophic factor (GDNF), BIO and B27, which are commercially available from a variety of distributors. Proliferating-enhancing amounts of these factors can vary depending on the species or strain of the cells, and the type of purity of the factors. Generally, 0.5 to 500 ng/ml of each factor within the culture solution is appropriate. In particular, the amount is between 4-50 ng/ml for GDNF, 1-10×103 units/ml for LIF, 0.5-5 μM BIO, and 1×B27. If basic fibroblast growth factor (bFGF) is used, e.g. for human and non-human primate cells, it is added in a quantity of 1-10 ng/ml. Typical combinations of growth factors are shown in Table 1 below.
Alternatively, or additionally, the culture may take place on a suitable feeder layer. Typical examples of feeder layer cells are embryonic fibroblasts derived from a mammal such as mouse, inactivated e.g. by mitomycin C-treatment or irradiation. Other conventional feeder layers may also be used, if desirable.
Suitable containers used for the culture of these cells may include, but are not limited to, conventional culture dishes, uncoated or coated with e.g. gelatine or Matrigel.
Optionally, the cells can be obtained from the culture by dissociating them with a suitable concentration of e.g. trypsin/EDTA or Collagenase IV, or any other commercially available substance such as TrypLE™ Select or TrypLE™ Express (Invitrogen) or Akutase or Dispase.
In a preferred embodiment, at most 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, in particular at most 30%, more particular at most 10%, of the ESC-like cells exhibit the c-kit antigen. The determination of the c-kit antigen expression may be performed using c-kit-specific antibodies in conventional FACS sorting in accordance with Examples 3.1 (mouse), 3.2 (human and non-human primate) given herein. The c-kit receptor (also known as CD117) is known to be expressed in differentiating spermatogonia and is listed, for example, in (Yoshinaga, K., et al., Development 113, 1991, 689-99; Vincent, S., et al., Development 125, 1998, 4585-93) under Accession Number NM021099; see also U.S. Pat. No. 6,949,364.
In a preferred embodiment, the mammalian testis cells are selected from the group consisting of human, non-human primate, mouse, bovine, pig, goat, rat, rabbit, ovine, horse, dog, cat and guinea-pig cells. The inventors show in the present application that they were able to successfully obtain ESC-like cells and products derived from these from three different species, i.e. human, non-human primate and mouse. Therefore, it is contemplated that the skilled person may also obtain similar results when using cells from other species such as listed above.
In a preferred embodiment, human or non-human primate cells are kept in a medium such as described above. Usually, the medium additionally contains one of the growth factors listed in Table 1 or any of the combinations of growth factors shown in the same table. In particular, all five of the growth factors listed in Table 1 are added to the medium. This medium may also be used in combination with a suitable feeder layer as defined above. Furthermore, when non-human primate cells are cultured, the medium may be DMEM further containing serum or serum replacement, as described above, and LIF and/or a feeder layer, as described above.
In another embodiment, the mammalian cells are of murine origin. Typically, the medium used herein is DMEM containing serum or serum replacement, as described above, particularly in the presence of LIF and/or a feeder layer, as described above. Preferably, the propagating takes place in the substantial absence of bFGF, EGF, and/or GDNF, in particular in the substantial absence of bFGF or EGF within the first 14 days, more particularly the first 7 days, most particularly the first 2-4 days of culture of the isolated mammalian testis cells. Thus, GDNF may be present during the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days of culture. The term “substantial absence”, as used herein, refers to a quantity of less than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 ng/ml bFGF or zero bFGF, less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 ng/ml EGF or zero EGF, and/or less than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 ng/ml GDNF or zero GDNF, in particular less than 0.5 ng/ml of each factor.
If desired, the propagating of the mammalian testicular cells takes place in the substantial absence of steel factor (SF) and/or Bone Morphogenic Protein 8 (BMP8). In particular, SF and/or BMP8 are present in a concentration of less than 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 12, 5, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5 ng/ml or zero SF and/or BMP8, more particularly less than 0.5 ng/ml of culture medium. The use of SF and BMP8 is described in U.S. Pat. No. 6,251,671. SF is a ligand to the c-kit receptor and is known to induce the differentiation of SSCs into meiotic and post-meiotic cells (Feng, L. X. et al., Science 2002, 297, 392-395). In another embodiment, prior to step a) of claim 1, the number of the isolated mammalian testicular cells can be increased, if desired, by propagating the cells in a suitable cell culture medium containing serum or serum replacement as described above, for a period of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 days. This step may be performed to further improve the quality and number of the isolated mammalian testicular cells, in particular if the testis biopsy should not provide sufficient material. Ideally, the step is carried out until SSC-colonies are observable in the culture which the skilled person will recognize as having reached a suitable size and number. As an example, suitable colonies may contain 20-100 cells and have a diameter of about 60-250 μm.
In another embodiment, the mammalian ESC-like cells are expanded after step (a) and before step (b) in the presence of a suitable feeder layer as described above and/or one or more suitable growth factors as described above for a period of 1-250, in particular 15-210, more particularly 30-210, most particularly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120 days. Usually, the ESC-like cells are passaged at a dilution of 1:3 to 1:6, typically every 1-4 or 1-7 days.
In a further embodiment, the mammalian testicular cells are provided as a preparation derived from an adult mammalian testis as described above. The preparation may be enriched for spermatogonial stem cells (SSCs) for example by a) selecting cells according to protein expression in the same cells or by b) use of a pre-plating method or by c) mechanical isolation of ESC-like colonies from the culture under microscope supervision. If step a) is chosen, examples for such proteins suitable for selection are surface proteins such as CD-9 or SSEA1 for murine cells, EpCAM and SSEA3 for human cells, or Integrin α6 or Integrin β1. The expression of such proteins may be monitored for example by antibody staining and FACS sorting. Another technique is the selection of cells by promoter-driven gene expression as described in example 1.1 below. An example for a pre-plating method as mentioned under b) above is also described among others in the examples 3.1, 5.1 following below. Such enrichment techniques have the potential to increase the number of SSCs obtained by 5-fold to 700-fold.
In another embodiment, the mammalian ESC-like cells express different levels of the antigens c-kit, Thy-1 and/or the gene Stra8. Sequences for the Thy-1 protein and the Stra8 gene can be found under Accession Number BC065559 for Thy-1 and NM 009292 for Stra8. The expression levels of the antigens c-kit and Thy-1 may be determined by techniques such as FACS sorting as described in the examples 3.1 and 3.2 described below. Typical expression levels of the antigens c-kit and Thy-1 found in ESC-like cells of three different species are displayed in the following Table 2. In a preferred embodiment, the ESC-like cells are characterized by the expression of Stra8 and/or c-kit. The c-kit expression may be exhibited by at most 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, in particular at most 30%, more particularly at most 10%, of the ESC-like cells. Expression of the gene Stra8 can be determined by procedures such as standard RT-PCR using suitable oligonucleotide primers such as described in example 4 below and subsequent analysis for example by subjecting the PCR probe to standard agarose gel electrophoresis. Expression of the gene Stra8 can be ascertained by the appearance of a visible band on such a gel.
In another embodiment, the mammalian ESC-like cells are capable of forming embryoid bodies as defined below.
In another aspect, the present invention relates to an ESC-like cell as obtainable by the process described above and in the exemplifying section. In particular, these cells are characterized by the expression of the gene Stra8 and/or the c-kit receptor, as outlined above.
In still another embodiment, the invention relates to a pharmaceutical preparation comprising the ESC-like cells and a pharmaceutically acceptable carrier. These pharmaceutical preparations contain ESC-like cells according to the invention in a quantity of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50% particularly 60 or 70%, more particularly 80 or 90% and most particularly 100%, relative to the total number of cells present in the preparation.
Another aspect of the invention relates to a method of producing embryoid bodies from ESC-like cells, comprising culturing the ESC-like cells according to the invention under conditions effective to yield embryoid bodies, and optionally recovering the embryoid bodies from the culture. The term “embryoid body”, as used herein, refers to clumps of cellular structures that contain tissue from all three germ layers: endoderm, mesoderm, and ectoderm. The typical morphology of embryoid bodies is shown in
In another embodiment, the invention relates to a pharmaceutical preparation comprising an embryoid body according to the invention and a pharmaceutically acceptable carrier.
In still another aspect the present invention relates to a method of producing a tissue and/or a differentiated cell, comprising culturing an ESC-like cell according to the invention and/or an embryoid body according to the invention under conditions effective to yield the tissue and/or the differentiated cell, and optionally recovering the tissue and/or the cell. For example, cardiomyocytes may be obtained via embryoid bodies, while neuronal cells may be obtainable directly from the ESC-like cell according to the invention. Depending on the desired cell or tissue type, the skilled person will select the appropriate factors and culture conditions suitable for obtaining the desired product as well as suitable tissue or cell-specific markers to verify that the desired products have been obtained. Examples for such tissue-specific markers are provided in the experimental section below in 5.1. In addition, it is contemplated that tissue-specific promoters driving a gene encoding for example antibiotics or fluorescent proteins are introduced by transfection of the cell culture and the desired differentiated cells are then selected by way of, e.g. FACS sorting or antibiotic selection. Examples for such promoters include the isl-1, Nkx2.5, MLC-2v or MHC promoter for the selection of cardiomyocytes, the promoters of myoD or myf5 for selection of myocytes, the promoter of α-fetoprotein or albumin for selection of hepatocytes, the promoter of nestin for selection of neural progenitor cells, the promoter of TH for selection of dopaminergic neurons, or the promoter of Nkx2.2, insulin or Nkx6.1 for selection of pancreatic cells. Methods of recovering cells from the culture include for example a mechanical selection such as shown in 5.1 of the experimental section.
In a preferred embodiment the desired tissue is selected from the group consisting of heart, vessels, neurons, skin, the eye, the nose, the ear, the spinal cord, a nerve, the trachea, the mouth, the esophagus, the small intestine, the large intestines, the ureter, the bladder, the urethra, a gland such as hypothalamus, pituitary, thyroid, pancreas and adrenal glands, the ovary, the oviduct, the uterus, the vagina, a mammary gland, the testes, the penis, a lymph node, a tendon, a ligament, brain, intestine, lung, muscle, stomach, liver, kidney, spleen, fat, bone, cartilage and epithelium. In another embodiment, the cell is selected from the group consisting of a cardiomyocyte, a skeletal muscle cell, a smooth muscle cell, an endothelial cell, a vascular cell, a vascular smooth muscle cell; a neural cell, a neuronal cell, in particular a neuron, more particularly a dopaminergic neuron, a cholinergic neuron, a GABAergic neuron, a serotoninergic neuron; a glial cell, a dendritic cell, a hepatocyte, a pancreatic cell, particularly a pancreatic hormone-producing cell; more particularly an insulin-expressing cell, a beta-cell, a glucagon-expressing cell, an alpha-cell, a somatostatin-expressing cell, a pancreatic polypeptide-expressing cell; a chondroicyte, a blastocyte, a astrocyte, a oligodendrocyte, a blood cell, a progenitor cell, a urogenital cell, a gastrointestinal cell, a glandular cell, an adiopocyte, an osteocyte, a microglia, an epithelial or epitheloid cell, and a bile duct cell.
In still a further embodiment the invention relates to a tissue and/or differentiated cell thus obtained. As set out above, the type of tissue and/or cell can be verified by use of tissue and/or cell-specific markers such as those identified in the experimental section herein below.
The invention also relates to a pharmaceutical preparation comprising a cell and/or tissue as outlined above and a pharmaceutically acceptable carrier. These pharmaceutical preparations contain cells or tissues according to the invention in a quantity of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 277, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50% particularly 60 or 70%, more particularly 80 or 90% and most particularly 100%, relative to the total number of cells present in the preparation.
In another aspect, the invention relates to the use of an ESC-like cell, an embryoid body, or a tissue or cell, or a pharmaceutical composition as outlines above for producing cell lines, tissues, organs, blastocysts, and transgenic animals. Examples for ways to obtain such cell lines are described in the examples below. It is apparent that such cell lines might be useful for studying diseases. The establishment of cell lines from humans or animals with genetic abnormalities or varieties could provide new options for studying the corresponding cellular phenotype under in vitro conditions. Examples for the use of blastocysts and the creation of transgenic animals are also provided in the examples below in 6.1 and 6.2 of the experimental section. It is contemplated that such transgenic animals would allow the development of various cell types for studying physiological or pathopysiological processes. In the light of present disclosure the skilled person can readily produce such transgenics.
In another embodiment, the use of an ESC-like cell, an embryoid body, a tissue or cell, or any of the pharmaceutical compositions described above is contemplated for the use in cell-based organ regeneration therapy, tissue-based organ regeneration therapy, and autologous or allogenic cell, tissue or organ transplantation. Such uses might allow the establishment of individual cell-based therapy, since the donor and recipient of the cell and/or tissue could be identical. The immunological profile of these cells might allow also allogenic transplantation. It is evident that by the use of such techniques, the ethical problems associated with human embryonic stem cells might be avoided.
In another embodiment, the use of an ESC-like cell, an embryoid body, a tissue or cell, or any of the pharmaceutical compositions outlined above is contemplated for the prevention and/or therapy of a disease or disorder such as chronic heart disease, Parkinson's disease, diabetes, liver failure, infertility and end-stage kidney disease, traumatic spinal cord injury, Purkinje cell degeneration, neural degeneration, Alzheimer's disease, Lewy Body Dementia, multiple sclerosis, duchenne's muscular dystrophy, muscular dystrophy, heart failure, osteogenesis imperfecta, osteoporosis, cancer, an autoimmune disease, a neurodegenerative disease, a respiratory disease, a vascular disease, a trauma, burn, head trauma, spinal cord injury, stroke, myocardial infarction, arthrosis, Huntington's disease, Tourette's syndrome, amytrophic lateral sclerosis, Addison's disease, pituitary insufficiency, liver failure, inflammatory arthrophathy, neuropathic pain, blindness, hearing loss, arthritis, a bacterial infection, a viral infection, a sexually transmitted disease and a damage of the skin, the eye, the nose, the ear, the brain, the spinal cord, a nerve, the trachea, the lungs, the mouth, the esophagus, the stomach, the liver, the small intestines, the large intestines, the kidney, the ureter, the bladder, the urethra, a gland such as hypothalamus, pituitary, thyroid, pancreas and adrenal glands, the ovary, the oviduct, the uterus, the vagina, a mammary gland, the testes, the penis, a lymph node, a vessel, the heart, a blood vessel, a skeletal muscle, a smooth muscle, a bone, cartilage, a tendon or a ligament. It is contemplated that, based on the present disclosure, the skilled person may readily use the ESC-like cells, embryoid bodies or pharmaceutical compositions of the invention to put the above-described medical applications into practice.
d-f, Epithelial cell/hepatocyte differentiation. d, Pan-cytokeratin-positive epithelial cells on day 14 after plating of EBs at day 5 (day 5+14). e, Cytokeratin 18-positive large epithelioid cells on day 5+17. f, Cytokeratin 7-positive bile duct cells on day 5+14. Scale bar, 25 μm (a-f). Nuclear staining with DAPI.
b, Genomic DNA isolated from different tissues of animal number 9, 19 and 5 were subjected to genomic PCR using LacZ-specific primers, revealing participation of SSC-derived cells in different tissues. 1, muscle; 2, lung; 3, kidney; 4, spleen; 5, intestine; 6, heart; 7, stomach; 8, testis; 9, liver; 10, brain. +: DNA from ROSA26 mice as positive control; −: DNA from wildtype mice as negative control.
The following examples are meant to further illustrate, but not limit, the invention. The examples comprise technical features and it will be appreciated that the invention relates also to combinations of the technical features presented in this exemplifying section.
For selection and enrichment of mouse SSCs, a spermatogonia-specific marker Stra8, which is expressed restrictedly to the male germ cells from E14.5 to spermatogonia (Oulad-Abdelghani, M. et al., J Cell Biol 135, 1996, 469-77), was used. The activity of its regulatory sequences enables the enrichment of germline stem cells in transgenic mice (Nayemia, K. et al., Hum Mol Genet 13, 2004, 1451-60). Mice from the transgenic mouse line Stra8-EGFP (FVB) (Nayemia, K. et al., Hum Mol Genet. 13, 2004, 1451-60) were crossed to Rosa26 mice (C57BL6/129Sv; The Jackson Laboratory) to generate the double transgenic mouse line Stra8-EGFP/Rosa26, which has a mixed FVB/C57BL6/129Sv genetic background and was used through the study. Stra8-EGFP8 and Stra8-EGFP/Rosa26 transgenic mouse lines showed specific Stra8-EGFP expression in spermatogonia of adult animals.
For isolation of spermatogonial stem cells (SSCs), testes of adult transgenic males (n=15 mice, 6-week-old) of mouse line Stra8-EGFP/ROSA26 were collected aseptically in serum-free Dulbecco's modified Eagle's medium (DMEM, Gibco/BRL). After decapsulation of the testes, interstitial cells were removed by mechanical agitation and washing with the same medium. The isolated seminiferous tubules were treated with collagenase and trypsin (Boehringer) as described previously (Nayemia, K., et al., Methods Mol Biol 253, 2004, 105-20). Testicular cells (2-3×107 per mouse) were cultured on 0.1% gelatine-coated culture dishes in medium containing 20% fetal calf serum (FCS, selected batch for mouse embryonic stem cell culture; Invitrogen), 1×non-essential amino acids (NEAA; Invitrogen), 2 mM L-glutamine (Invitrogen), 50 μM β-mercaptoethanol (β-ME; Promega), 1× Pen/Strep (PAN) and 4 ng/ml glial-derived neurotrophic factors (GDNF; Tebu) for 4-7 days. Instead of traditional DMEM, KnockOut™ DMEM (Invitrogen) can be used. At the same time, instead of FCS, KnockOut™ serum replacement can be used.
GFP positive cells (1-2×106) were separated by fluorescence-activated cell sorting (FACS) using FACS Vantage cell sorter (Becton Dickinson) equipped with 488-nm argon and 633-nm He—Ne lasers. In all sorting procedures, an initial gating was performed in order to exclude cell debris and cell doublets based on forward- and side-scatter information, and the system was first calibrated with wildtype tissue in order to determine the background signal for GFP fluorescence. Green fluorescence was measured through a 530-nm/30-nm bandpass filter after illumination with the 488-nm line of an argon ion laser. The sorted GFP+ cells were pelleted and cultured on 0.1% gelatine-coated culture dishes in medium containing 15% FCS, 1×NEAA, 2 mM L-glutamine, 50 μM β-ME and 4 ng/ml GDNF for 1 week.
Transplantation of Murine SSCs into Seminiferous Tubules of Testes
At 6-8 weeks of age, the recipient mice were injected i.p. with busulfan (40 mg/kg body weight), which destroys endogenous spermatogenesis. Recipients were used for transplantation at 4 weeks after injection. To prove stem cell activity of FACS-sorted GFP+-cells, approximately 1-3×105 FACS-sorted GFP+ cells were transplanted via rete testis into seminiferous tubules of one testis (Nayernia, K., et al., Methods Mol Biol 253, 2004, 105-20). As external control, busulfan treated non-transplanted males were used. After 20 weeks, recipient mice were killed and testes were examined by LacZ staining as described (Patapoutian, A., et al., Development 118, 1993, 61-9).
After 5-6 months, regeneration of normal spermatogenesis was observed by LacZ+ cells (data not shown). These results indicate that GFP+ cells fulfil the criteria of SSCs and are not derived from testicular teratomas. Testicular teratomas are rare in most inbred mouse strains and occur spontaneously at a rate of 1-5% in 129/Sv inbred strains (Stevens, L. C. & Hummel, K. P., J Natl Cancer Inst 18, 1957, 719-47). In a mixed C57BL/6 and 129/Sv genetic background, a lower number of tumour suppressor gene TRP53-deficient mice develop testicular tumours than on a pure 129/Sv genetic background (Donehower, L. A. et al., Mol Carcinog 14, 1995, 16-22). We used the transgenic mouse line which has a mixed FVB/C57BL6/129Sv genetic background. We did not observe testicular tumours out of 15 males in this mixed genetic background.
SSCs from adult mice with three different genotypes (C57BL/6, 129/Ola and FVB) were also isolated without the genetic selection. The testicular cells were first cultured in DMEM containing 20% FCS, 1×NEAA, 2 mM L-glutamine, 50 μM β-ME, 1× Pen/Strep and 4 ng/ml GDNF on 0.1% gelatine-coated culture dishes at 37° C. and 5% CO2 overnight. After removal of attached cells, spermatogonia, which remained in suspension, were collected and cultured on fresh gelatine-coated tissue culture dishes in the same medium for 1-2 weeks.
1.2. Isolation of Spermatogonial Stem Cells from Non-Human Primate and Human
For isolation of spermatogonial stem cells from adult non-human primate, approximately 20 g of testicular tissue was used for each cell isolation procedure. After decapsulation, the testes were first washed in DPBS, minced into small pieces and suspended in DMEM supplemented with penicillin and spreptomycin.
For isolation of human SSCs, testis biopsies obtained from patients (adult male) was first washed in Dulbecco's phosphate buffered salt solution (DPBS) and then minced into small pieces and suspended in DMEM supplemented with penicillin and spreptomycin.
The testicular tissues were then treated with one of the two following methods.
Method I: The minced testicular tissues were incubated in Hanks' Balanced Salt Solution (HBSS) containing 1 mg/ml collagenase for 15-30 min at 37° C. After centrifugation for 5 min at 1000 rpm, the pellets were washed three times in pure HBSS medium, and resuspended in 5 ml Trypsin/EDTA (1×Trypsin/EDTA, 0.05%/0.02% solved in DPBS). After repeated pipetting, 10-20% vol. FCS was added. The large tissue residues were removed with sterile tips, and small residues of tissue were removed by filtrating through 70 μm nylon filters (BD Falcon Cell Strainer).
Method II: Minced pieces were incubated in DMEM containing 1 mg/ml collagenase, 1 mg/ml hyaluronidase, 1 mg/ml trypsin and 5 μg/ml DNase at 32° C. for 60 min in a shaking water bath. After three washes in DMEM and removal of most of interstitial cells, seminiferous tubular fragments were incubated in DMEM containing 1 mg/ml collagenase, 1 mg/ml hyaluronidase, and 5 μg/ml DNase at 32° C. for 45 min. After repeated pipetting, tubular fragments were centrifuged at 30×g for 2 min. Cells in the supernatant were collected, and filtered through 70 μm nylon filters.
The flow through was centrifuged for 5 min at 1000 rpm. The resulting pellet was resuspended in DMEM/F12 medium (Invitrogen) containing 20% FCS, 1×NEAA, 2 mM L-glutamine, 50 μM β-ME, 1× Pen/Strep, 4 ng/ml bFGF (Tebu) and 4 ng/ml GDNF, and incubated on 0.1% gelatine-coated culture dishes at 37° C. and 5% CO2 overnight. After removal of attached cells, spermatogonia, which remained in suspension, were collected and cultured on fresh gelatine-coated tissue culture dishes in the same medium for 3-10 days.
The Stra8-EGFP+ cells that had been tested for the contribution of spermatogenesis were used to establish the conditions required to convert these cells into pluripotent stem cells. To maintain the GFP+ cells at undifferentiated state, four different culture conditions were tested. Condition I: Cells were cultured in gelatine-coated culture dishes with basic medium, DMEM (high glucose formulation; Gibco/BRL) supplemented with 15% FCS, 2 mM L-glutamine, 50 μM β-ME, 1×NEAA. Condition II: Cells were cultured in gelatine-coated culture dishes with basic medium containing 103 units/ml leukaemia inhibitory factors (LIF; ESGRO, Chemicon). Condition III: Cells were cultured on a feeder layer of mitomycin C-inactivated mouse embryonic fibroblasts with basic medium. Condition IV: Cells were cultured on a feeder layer with basic medium containing 103 units/ml LIF (=embryonic stem cell culture condition). Standard embryonic stem cell (ESC) culture condition as described (Guan, K., et al., Cytotechnology 30, 1999, 211-26) was used for cultivation of mouse MPI-II ESCs isolated from inner cell mass of the blastocysts (Voss, A. K., et al., Exp Cell Res 230, 1997, 45-9). Instead of traditional DMEM, KnockOut™ DMEM (Invitrogen) can be used. At the same time, instead of FCS, KnockOut™ serum replacement can be used.
The GFP+SSCs were first propagated in basic medium (condition I) containing 4 ng/ml GDNF for 2 weeks. The cells were dissociated by trysin/EDTA solution (0.2% Trysin/0.02% EDTA=1:1 in PBS) and passaged at a dilution of 1:3 every 4-5 days. Afterwards, the cells were cultured in four different conditions. When the GFP+SSCs were cultured under condition I, the majority of the colonies that formed were similar to cultured epiblast cells (
Pre-Plating: briefly, the testicular cells were harvested from the tissue culture dishes by treatment with 0.1% trypsin and 0.01% EDTA in phosphate-buffered saline (PBS). The dissociated cells were replated in 0.1% gelatine-coated culture dishes and put back to the incubator. The Sertoli, fibroblasts and endothelial cells attached to the culture dishes after about 1.5 hours. The floating cells were collected and transferred to a fresh 0.1% gelatine-coated tissue culture dish. ES-like cells (
Mouse SSC cultures indicated that both feeder layer and LIF are preferable for the establishment of ESC-like cells. Therefore, culture conditions II to IV were applied, which had been used for the cultivation of mouse ESC-like cells derived from SSCs, for the establishment of human and non-human primate ESC-like cells. Human as well as non-human primate SSCs were dissociated by trypsin/EDTA solution (0.2% Trypsin/0.02% EDTA=1:1 in PBS) and passaged at a dilution of 1:3 every 5-7 days. ESC-like colonies derived from non-human primate SSCs appeared after 2-3 passages (ca. two-week culture;
To optimize the culture condition, DMEM/F12 (1:1; Invitrogen) was applied instead of DMEM as basic medium and several growth factors for long-term culture were added: GDNF (4-50 ng/ml), bFGF (1-10 ng/ml), LIF (103 Units/ml), BIO (Calbiochem) and 1×B27 (Invitrogen). Collagenase IV (1 mg/ml in PBS) was used instead of trypsin/EDTA for passaging the cells. First, it was found that DMEM/F12 instead of DMEM as basic medium resulted in better proliferation of human as well as non-human primate SSCs. Tightly packed ESC-like colonies derived from human SSCs after 7 passages (about 1 month;
For characterization by flow cytometry, the SSCs as well as ESCs were harvested from the tissue culture dishes by treatment with 0.1% trypsin and 0.01% EDTA in phosphate-buffered saline (PBS). To eliminate the feeder cells, pre-plating method was applied. Briefly, the harvested cells were replated in 0.1% gelatine-coated culture dishes and put back to the incubator. The feeder cells attached to the culture dishes after about 1.5 hours. The floating cells were collected and fixed in 4% paraformaldehyde. The fixed cells were washed twice with two volumes of PBS and then blocked with 1% BSA at RT for 1 hour. The cells, in solution at a concentration of 0.5×106 cells/ml, were stained for 30 min with an empirically determined amount of each antibody. Labelled cells were thoroughly washed with two volumes of PBS. The following primary antibodies were used: mouse anti-SSEA-1 (clone MC480, Developmental Studies Hybridoma Bank, DSHB), mouse anti-SSEA-3 (clone MC631, DSHB), mouse anti-SSEA-4 (clone MC893-70, DSHB), mouse anti-Oct4 (clone 9E3.2, Chemicon), phycoerythrin (PE)-conjugated anti-mouse c-kit, Thy1, Ter119, Sca-1 and CD34 (Becton Dickinson). Cy3-conjugated goat anti-mouse IgG+IgM was used as secondary antibodies. The labelled cells were analyzed on a FACScan (Becton-Dickinson) by collecting 50,000 events with the Cell Quest software program (Becton-Dickinson).
For characterization by immunocytochemistry, cultured SSCs were subjected to specific immunostaining for SSEA-1 (mouse monoclonal antibody, clone MC480, Developmental Hybridoma Bank), Oct4 (mouse monoclonal antibody, clone 9E3.2, Chemicon), SSEA-3 (mouse monoclonal antibody, clone MC631, Developmental Hybridoma Bank), and GFP (rabbit polyclonal antibody, abcam) using the procedure as described (Wobus, A. M. et al., Methods Mol Biol 185, 2002, 127-56).
Characterization of cultured SSCs by ALP staining was carried out using a Sigma alkaline phosphatase substrate kit (86C-1 KIT) according to manufacturer's protocol.
Results: The phenotype of cultured GFP+SSCs is dependent on different culture conditions (
For characterization by flow cytometry, the cultured non-human primate and human were harvested from the tissue culture dishes by treatment with 0.1% trypsin and 0.01% EDTA in phosphate-buffered saline (PBS). The dissociated cells, in solution at a concentration of 0.5×106 cells per ml, were stained for 30 min with an empirically determined amount of each antibody. Labelled cells were thoroughly washed with two volumes of PBS. The following primary antibodies were used: phycoerythrin (PE)-conjugated anti-human CD9, and CD117 (c-kit), and FITC-conjugated anti-human CD90 (Thy1) and CD49f (Integrin α6) (Becton Dickinson). PE-IgG1 and FITC-IgG1 was used as negative control. The labelled cells were analyzed on a FACScan (Becton-Dickinson) by collecting 50,000 events with the Cell Quest software program (Becton-Dickinson). Non-human primate SSCs cultured in DMEM/F12 medium containing 15% FCS, 2 mM glutamine, 1×NEAA and 50 μM β-ME are partially positive for CD90 (Thy-1), CD9 and CD49f (integrin α6), but negative for CD117 (c-kit) (
Mouse SSCs cultured with different conditions (I-IV) and ESC-like cells were collected for total RNA isolation (Promega). 150 ng of DNase-treated RNA was used for first-strand cDNA synthesis. One-tenth of the cDNA reaction was taken as PCR template and amplified for 25-35 cycles. GAPDH was used as an internal control. Stem cell-specific maker genes were analysed.
Results: The RT-PCR analysis showed that the cultured cells under all four conditions, like ESCs, expressed genes encoding transcription factors Oct3/4, nanog, UTF-1, Esg-1 and REX-1 (
The cultures derived from wildtype mice of three different strains (C57BL6, FVB and 129/Ola), similar to GFP+ cells, express Stra8, Oct3/4, nanog, UTF-1, Esg-1 and REX-1 and are susceptible to LIF (
Non-human primate and human SSCs cultured with different conditions were collected for total RNA isolation (Promega). 150 ng of DNase-treated RNA was used for first-strand cDNA synthesis. One-tenth of the cDNA reaction was taken as PCR template and amplified for 25-35 cycles. GAPDH was used as an internal control. Stem cell-specific maker genes were analysed.
Results: The RT-PCR analysis showed that the cultured cells under all four conditions, like ESCs, expressed genes encoding transcription factors Oct3/4, nanog (
5. In Vitro Differentiation and Characterization of maGSCs
5.1. In Vitro Differentiation and Characterization of Murine maGSCs
For differentiation into ectodermal, mesodermal and endodermal lineages, mouse maGSCs were cultivated as embryoid bodies (EBs) (
Results: An overall decrease in GFP+ cell populations was observed upon EB differentiation (
Differentiation of mesodermal lineages (for example, cardiac, skeletal muscle, and vascular cells) was confirmed by the expression of the early mesoderm marker Brachyury (T) gene (King, T., et al., Mech Dev 79, 1998, 29-37) as well as lineage-specific genes and proteins. The expression of T gene was maximal at early differentiation stages (
In addition, maGSCs derived from wildtype mice of three different strains (C57BL6, FVB and 129/Ola) can also spontaneously differentiate into cardiac cells by inducing EB formation (
Cardiomyocyte phenotype of these contracting areas can be demonstrated by immunostaining of proteins relevant for myocyte contraction. For a better demonstration of the structural organization of intracellular, especially sarcomeric proteins, cardiomyocytes are isolated as single cells from the beating areas of EBs (at day 5+4) mechanically using a micro-scalpel under an inverted microscope (Wobus, A. M. et al., Methods Mol Biol 185, 2002, 127-56). Tissues were collected in Tyrode's solution with (in mM) 140 NaCl, 4 KCl, 1 MgCl2, 10 glucose, 5 HEPES (pH 7.4 with NaOH). The isolated clusters were digested in collagenase type 2 (310 U/ml, Worthington) supplemented Tyrode's solution with 30 μM CaCl2 at 37° C. for 30 minutes. For the isolation of small cardiac clusters, the incubation time was shortened to 15 minutes. The cells were then resuspended and dissociated in 100 μl of KB medium at 37° C. for 30 minutes. The cell suspensions were transferred into tissue culture plates containing gelatin-coated cover slips and incubated in differentiation medium at 37° C. overnight. Cardiomyocytes began rhythmical contractions after 24 hours and were used for immunostaining 3 days later.
Isolated cardiomyocytes were analysed by specific immunostaining for sarcomeric α-actinin (rabbit polyclonal antibody, clone 653, kindly from Prof. DO Fürst, University of Potsdam, Germany), α-myosin heavy chain (mouse monoclonal antibody, clone MF20, Developmental Hybridoma Bank), cardiac troponin T (Ab-1, mouse monoclonal antibody, clone 13-11, Lab Vision). Accordingly, single cardiomyocytes isolated from beating areas exhibit sarcomeric striations when stained with α-sarcomeric actinin (
In cardiac muscle, where Ca2+ influx across the sarcolemma is essential for contraction, the dihydropyridine (DHP)-sensitive L-type calcium channel (DHP receptor) represents the major entry pathway of extracellular Ca2+. Using immunofluorecence staining with a specific α1c antibody we could show that α1 subunit of L-type calcium channels are expressed in cardiomyocytes at day 5+6 in a striated pattern (
To analyse the cell-to-cell coupling in the synchronously contracting cardiac clusters, a double-staining in the cells with either pan-cadherin (mouse monoclonal antibody, clone CH-19, Sigma) and α-actintin or connexin 43 (Cx43; monoclonal antibody, clone 4E6.2, Chemicon) and α-actintin was performed. It was found that pan-cadherin localized at cell-cell junctions indicating the presence of adhering junctions between cardiac cells (
Functional coupling between cells is confirmed by fluorescence recovery after photobleaching (FRAP) analysis. Briefly, cardiac clusters were loaded with the membrane-permeant fluorescent dye calcein AM (5 μmol/L; Invitrogen) in Ca2+-free Tyrode solution for 20 min at 37° C. After washing away the excess extracellular fluorescent dye to prevent further loading, the cultures were bathed in Ca2+-free Tyrode solution and placed on the stage of a Zeiss LSM 5 PASCAL laser-scanning confocal microscope. Using Zeiss software, a rectangular region encompassing 30-50% of a single cell within a cell cluster was selected and its fluorescence was bleached by a high-intensity laser pulse (488 nm, 3-5 s duration). This caused immediate loss of calcein fluorescence emission recorded through a 505 nm long-pass emission filter. Calcein redistribution from adjacent unbleached cells through connexin pores into the bleached region of interest (ROI) was recorded in subsequent confocal images acquired at 30 s intervals for up to 10 min using a low-intensity laser pulse. Fluorescence recovery within ROI was plotted as a function of time and fit to a single exponential function: IROI (t)=A [1−exp (−kt)], where IROI (t) is the ROI fluorescence intensity at time t, A is the amplitude of fluorescence recovery, and k is the rate of recovery. The latter is considered a measure of gap junction permeability. For the inhibitor studies, the cells were incubated in Ca2+-free Tyrode solution containing the gap junction uncoupler 3β-hydroxy-11-oxoolean-12-en-30-oic acid 3-hemisuccinate (carbenoxolone, 50 μmol/L; Sigma).
Fluorescence recovery of a gap junction permeable dye (calcein AM) is observed consistently for cardiac cells within 5 minutes after photobleaching (
In mature cardiac cells, depolarization of the cell membrane during the action potential activates L-type Ca2+-channels leading to Ca2+ influx and subsequent release of Ca2+ from intracellular calcium stores. To characterize whether mouse SSC-derived cardiomyocytes could enter a fully differentiated cardiac phenotype, the dissociated cardiomyocytes were analysed by patch-clamp electrophysiology. Mouse SSC-derived cardiomyocytes show spontaneous action potentials (APs,
To determine the functional expression of α-adrenergic receptors in cardiomyocytes, the effects of β-adrenergic agonist isoproterenol (Iso; 1 μmol/L) on APs were studied. It is shown that the AP frequency was significantly increased from 0.80±0.15 Hz to 1.62±0.21 Hz (n=5; P<0.05) in response to Iso stimulation (
Intracellular Ca2+ was assessed in SSC-derived cardiomyocytes at day 5+7 using confocal microscopy. A typical triangle-shaped cardiomyocytes is presented in
The cardiac RyR2 serves as the major SR calcium-release channel to mediate the rapid rise of cytosolic free calcium. We find that the gene encoding RyR2 is expressed during cardiac differentiation of mouse maGSCs (
The differential expression of cardiac and skeletal muscle-specific transcription factors makes it possible to distinguish maGSC-derived cardiomyocytes from maGSC derived skeletal muscle cells. During EB differentiation, genes encoding the myogenic regulatory factors MyoD and Myf5 are strongly expressed from day 5+8 (
Vascular cells (endothelial and smooth muscle cells), which are essential for maintaining cardiac integrity and function in vivo can also be differentiated spontaneously from maGSC-derived EBs. The genes encoding smooth muscle α-actin (SM-α-actin), vascular smooth muscle MHC (VSM-MHC), platelet/endothelial-cell adhesion molecule 1 (PECAM-1), vascular endothelial growth factor receptor 2 (also known as flk-1) and von Willebrand factor (vWF) were expressed during EB differentiation (
The neuroectoderm differentiation in the maGSC-derived EBs was confirmed by the gene expression of nestin, a marker for neuroepithelial precursors, and genes expressed in differentiated neurons, such as synaptophysin and TH (
Epithelial-like cells found in all EB outgrowths are characterized using antibody anti-Pan cytokeratin (clone C-11, Sigma) which reacts with a wide variety of epithelial tissues (
For lineage-selective differentiation of maGSCs into neural cells, neural precursor differentiation from maGSCs in serum-free adherent monoculture was first induced. To start monolayer differentiation, 3×105 maGSCs in 3 ml standard ESC medium containing LIF were plated per 60 mm 0.1% gelatine-coated tissue culture dish (Nunc). Cells were maintained overnight, attached the culture dish and then switched to neural stem cell (NSC) medium (DMEM/F12 medium containing 2 mM glutamine, 1×N2 (Invitrogen), 1×NEAA, β-ME, 20 ng/ml bFGF and 10 ng/ml EGF) to start differentiation for 6 days. Medium is renewed every other day. Cells were then replated onto fresh tissue culture dishes in NSC medium containing 2% FCS, maintained overnight and then switch to NSC medium without FCS. After further cultivation for 4 days, cells were fixed and immunostained with antibodies against nestin (
To induce the generation of TH-positive cells, cells were resuspented in N2B27-I medium containing 20 ng/ml bFGF, 400 ng/ml sonic hedgehog (R&D) and 100 ng/ml FGF8 (R&D) and replating onto PDL-laminin-coated tissue culture dishes. Two days later, medium is changed to N2B27-I plus 20 ng/ml bFGF. Medium is then renewed every 2-3 days. High number of TH-positive cells among βIII-tublin-positive neurons were detected (
To induce pancreatic differentiation of maGSCs, two protocols used for ESC differentiation were applied. The differentiation protocol I was adapted from Blyszczuk et al. (
The differentiation protocol II was adapted from Lumelsky et al. (
For protocol I, 5-day-old EBs were plated in IMDM differentiation medium and EB outgrowths were dissociated into small clusters and replated at day 5+9 when cardiomyocytes, smooth muscle cells, endothelial and epithelial cells were detected according to their characteristic morphology (data not shown). Cell clusters were then cultured in pancreatic differentiation medium (serum-free N2 medium supplemented with NA). For protocol II, 4-day-old EBs were plated in ITSFn medium and nestin-positive cells were selected in serum-free medium.
RT-PCR analyses showed that genes encoding transcription factors that exert important functions in the control of pancreatic differentiation as well as genes encoding proteins that are normally present in pancreas were expressed in a developmentally controlled pattern (
To answer the question whether nestin is involved in pancreatic differentiation of maGSCs in vitro, co-localization of nestin and Pdx1 (using polyclonal antibody against Pdx1, Absam;
To investigate whether pancreatic hormone-producing cells could be generated from mouse maGSCs, immunofluorescence analyses were performed. At the terminal differentiation stage in protocol I, insulin-expressing islet-like clusters (using polyclonal antibody against insulin, Dako) could be demonstrated (
Furthermore, cells expressing glucagon (using polyclonal antibody against glucagon, Dako), somatostatin (using polyclonal antibody against somatostatin, Dako) and pancreatic polypeptide (using polyclonal antibody against pancreatic polypeptide, Linco;
Action Potential Measurements in Cardiomyocytes Derived from Mouse EBs:
The membrane potential (Em) of cardiomyocytes was measured at room temperature with ruptured-patch whole cell current clamp (Maier, L. S., et al., Circ Res 2003, 92, 904-11). Fire-polished glass microelectrodes of >10 MΩ resistance when filled with pipette solution were used. Pipette solution contained (in mM) 120 potassium aspartate, 8 KCl, 7 NaCl, 1 MgCl2, 10 HEPES, 5 Mg-ATP, 0.3 Li-GTP (pH 7.2 with KOH). A normal Tyrode's solution with 2 mM CaCl2 served as bath solution. Access resistance was typically ˜20 MΩ after patch rupture. Liquid junction potentials were corrected before recording started. Spontaneous action potentials (APs) were recorded 1 min after patch rupture and followed for about 2 min. Signals were acquired at 1 kHz, filtered with 2.9 and 10 kHz Bessel filters, and recorded with an EPC10 amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany). Action potentials were averaged from 10 single APs and analysis was done to determine the maximum rate of rise of the AP upstroke (dV/dtmax), AP amplitude (APA), AP duration at 90% of repolarisation (APD 90) and the maximum diastolic potential (MDP).
Intracellular calcium ([Ca2+]i) signals were recorded after incubating cells with 10 μM fluo-4 acetoxymethylester (Mobitec) for 15 min on a laser scanning confocal microscope (Zeiss LSM 5 PASCAL) (Maier, L. S., et al., Circ Res 2003, 92, 904-11). Cells were washed with Tyrode's solution as described above. Fluo-4 was excited via an argon laser (488 nm; 30 mW) and emitted fluorescence (F) was collected through a 505 nm long-pass emission filter. Changes in fluo-4 fluorescence (indicating fluctuation in cytosolic Ca2+) were recorded in frame and line scan mode while the cells were beating spontaneously. The images were acquired and analyzed using Zeiss software and fluorescence signals were normalized to basal cell fluorescence after fluo-4 loading (F0). Intracellular Ca2+ was assessed using line scan modus and calibrated by the following pseudo-ratio equation [Ca]i=Kd(F/F0)/(Kd/[Ca]i-rest+1−F/F0) with Kd=1100 nM and [Ca]i-rest=100 nM.
5.2. Formation of EBs from Non-Human Primate and Human SSCs In Vitro
For differentiation in vitro, non-human primate and human SSCs were first cultivated as embryoid bodies (EBs) using hanging drop method as described for mouse maGSCs. However, we found that this method is not suitable for EB formation of non-human primate and human SSCs because of their slow proliferation rate. When the non-human primate and human SSCs were cultivated as mass culture in IMDM supplemented with 20% FCS, 2 mM L-glutamine, 1×NEAA and 50 μM β-ME, 3D aggregates were formed (
6. In Vivo Differentiation of Murine maGSCs
To confirm the pluripotency of maGSCs in vivo, maGSCs (2×106 cells per injection) were subcutaneously injected into 8-week-old male SCID-beige mice (ten mice total) for teratoma formation. Six weeks after injection the mice were sacrificed and the resulting teratomas were examined histologically.
Results: The transplanted cells formed mature teratomas in all recipients (ten of ten) by 6 weeks after inoculation. The teratomas contained derivatives of three embryonic germ layers, including epithelium with intestinal differentiation (endoderm), striated muscle, smooth muscle, fat, bone and cartilage (mesoderm); and neural tissue (ectoderm) (
To determine the developmental potential of SSCs, SSCs were injected into early blastocysts. Wild-type blastocysts (3.5 dpc) were collected from the uteri of superovulated females by flushing with M2 medium (Sigma-Aldrich). Superovulation was induced by injection of 7.5 IU pregnant mare serum gonadotropin followed by injection of 7.5 IU human chorionic gonadotropin after a 48-hour interval. The collected blastocysts were washed with and cultivated in M16 (Sigma-Aldrich) under 5% CO2 in air at 39° C. Blastocyst injection was carried out by injecting GFP+ cells using standard procedures (Kimura, Y. and Yanagimachi, R., Development 1995, 121, 2397-405). 10-15 SSCs from Stra8-EGFP/Rosa26 mice were microinjected into 3.5-day-old blastocysts of C57BL/6 mice. Sixty-five injected blastocysts were transferred into the uterus of pseudopregnant mice. The number of litters born (42 animals) and animals per litter (6-7) were in line with the birth rate seen with ESCs (Schoonjans, L. et al., Stem Cells 21, 2003, 90-7). Animals born from microinjected blastocysts were of similar size as normal animals and did not display overt abnormalities. Chimeras were identified by extraction of genomic DNA from tail biopsies and subsequent PCR analysis using LacZ specific primers. Chimaerism could be detected in 39 of 42 of mice (˜93%) by LacZ PCR of 4-week-old animals. LacZ PCR analysis of genomic DNA isolated from different tissues of three negative animals revealed LacZ positive in some tissues (data not shown) indicating that in these animals SSCs also contributed to some somatic tissues. Chimaeric animals that had LacZ+ cells, as determined by PCR in the tail clip analysis, were killed at 10-20 weeks. A contribution of LacZ+SSCs to many tissues, including heart, brain, intestine, lung, skeletal muscle, liver, kidney, spleen, and testis was found by PCR analysis (
Human SSCs or maGSCs derived from SSCs isolated from testicular biopsies may have great potential for cell-based organ regeneration therapy. It is believed that these cells are pluripotent and are able to differentiate into derivatives of all three embryonic germ layers. The SSCs as well as SSC-derived differentiated cells might be used for cell-based organ regeneration therapy. Although SSCs cells are pluripotent like ESCs, use of these cells as well as their derivatives for transplantation will allow establishment of individual cell-based therapy, because the donor and recipient can be identical. If immunological profile of these cells allows allogenic transplantation needs to be explored. Furthermore, the ethical problem associated with human ESCs is avoided. In addition to cell transplantation strategies, our discovery provides new possibilities to study diseases. The establishment of maGSCs from transgenic animals would allow developing various cell types in vitro for studying physiological or pathophysiological processes. Moreover, the establishment of cell lines from humans with genetic abnormalities or varieties would provide new options for studying the corresponding cellular phenotype under in vitro condition.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP06/10529 | 11/2/2006 | WO | 00 | 3/11/2009 |
| Number | Date | Country | |
|---|---|---|---|
| 60732132 | Nov 2005 | US |
