Title of Invention	METHOD FOR ISOLATING PROGENITOR CELLS FROM MAMMARY SECRETION
Abstract	The present invention relates to a method for isolating progenitor cells from a human body, inclusive of all cells with stem cell-like characteristics, in particular pluripotent or multipotent progenitor cells, wherein such cells are directly or indirectly derived from human mammary secretion, be it colostrum, mature milk, or dry period secretion from males or females, of said human body during at least one of the following periods: non-pregnant period, pregnant period, lactating period, involuting period. The present invention furthermore relates to preferred uses of such isolated cells.

Title of Invention

METHOD FOR ISOLATING PROGENITOR CELLS FROM MAMMARY SECRETION

Abstract

The present invention relates to a method for isolating progenitor cells from a human body, inclusive of all cells with stem cell-like characteristics, in particular pluripotent or multipotent progenitor cells, wherein such cells are directly or indirectly derived from human mammary secretion, be it colostrum, mature milk, or dry period secretion from males or females, of said human body during at least one of the following periods: non-pregnant period, pregnant period, lactating period, involuting period. The present invention furthermore relates to preferred uses of such isolated cells.

Full Text	WO 2005/061696 PCT/CH2O04/00O73 1 SPECIFICATION TITLE Method for isolating cells from mammary secretion TECHNICAL FIELD The present invention relates to a method for isolating cells from a human body, as well as uses of such cells. BACKGROUND OF THE INVENTION Stem cells are defined as clonogenic, self-renewing progenitor cells that can generate a wide variety of more specialised cell types via the process of differentiation. Classically, it has been believed that there are two distinct types of stem cell. Embryonic stem (ES) cells are derived from the inner mass of the blastocyst, are pluripotent and thus are capable of generating into all differentiated cell types within the body. The other sub-population of stem cells are derived from ES cells and arc organ-or tissue-specific. These multipotcnt cells, also known as adult stem cells, were believed to be able to differentiate only into tissues from their organ of origin. An example of these multipotcnt cells are haematopoietic stem cells, which serve to continually regenerate the cells of the blood and immune system. Stem cells have been isolated from a wide range of tissues, from those that have a high rate of ongoing cellular turnover, such as blood, cord blood, bone marrow, skin, intestine, and breast tissue, to those with a low turnover such as brain, skeletal muscle. WO 2005/061696 PCT/CH20O4/000738 2 and juvenile teeth. Irrespective of the tissue of origin, a long standing dogma has been that adult stem cells can only differentiate into the tissues from which they were derived. However recent work has demonstrated that upon exposure to a novel environment, organ-specific stem cells can overcome these intrinsic restrictions to transdifferentiatc into other tissues. For example it has been shown that neural stem cells can transdiffercntiate into blood cells, bone-manow derived stem cells can transdifferentiate into muscle, brain, liver and heart cells, and skin derived stem cells can transdifferentiate into brain cells. Therefore it now appears likely that the dogma associated with developmental restriction of organ-specific stem cells is incorrect and it is feasible that these ES cells under appropriate environmental stimuli, can transdifferentiatc into another cell type. Human milk contains a mixture of different cell types. Secretory epithelial cells (lactocytes) are found in milk due to them sloughing off the basement membrane of the breast as a consequence of the pressure associated with the continued filling and emptying of the breast. Lactocytes account for approximately 10-20% of the total cell population. The majority of the remainder of cells found in human milk are leukocytes (immune cells such as lymphocytes, macrophages, monocytes, natural killer cells, basophils, cosinophils, and neutrophils), and are believed to be in milk to both protect the breast from infection and to provide immune protection for the infant. To date, these arc the only cell types believed to be contained in milk. SUMMARY OF THE INVENTION The objective problem underlying the present invention is therefore to provide a new method for isolating progenitor cells from the human body. In this context, the term progenitor cells shall include all cells with stem cell-like characteristics, preferentially but not exclusively including pluripotent or multipotent progenitor cells like for example stem cells. The present invention solves the above problem by deriving such cells directly or indirectly from human mammary secretion, be it colostrum, mature milk, or dry period WO 2005/061696 PCT/CH 2004/000738 3 secretion from males or females, of said human body during at least one of the following periods: non-pregnant period, pregnant period, lactating period, involuting period. In other words, here we demonstrate that surprisingly, progenitor cells can also be found in human lactation milk and that these cells have the potential to be utilised for the generation of tissues for the mother and infant. It has to be noted that not only human mammary secretion but generally mammary secretion from mammalian species can be used for the isolation of corresponding progenitor cells. It can unambiguously be shown by means of progenitor-cell-specific antibodies, that indeed mammalian secretion, i.e. for example human milk, comprises progenitor cells. In a first preferred embodiment of the present invention, said progenitor cells are isolated from the mammary secretion in that non-cellular parts of the mammary secretion are separated from the cellular parts and that in particular non-pluripotcnt or non-multipotent cells are removed from cellular parts thus derived. The cellular parts of mammary secretion, apart from pluripotent cells may further comprise secretory epithelial cells, leucocytes and in particular nonhuman cells like bacterial cells. Those non-pluripotent cells are preferentially removed from the mammary secretion. According to another preferred embodiment of the present invention, the mammary secretion during lactating periods is used for the isolation of the progenitor cells, wherein the mammary secretion during particular stages of mammary secretion such as: after beginning of individual feeding; versus end of individual feeding; lactation phase; preferably early lactation, is used. A particularly useful and practicable way of isolating those progenitor cells from the secretion is possible if magnet beads are used. Those magnetic beads are to this end preferentially connected to progenitor-cell-specific antibodies allowing the attachment of the beads to the progenitor cells. Typically, in a first step cellular components are washed out of the mammary secretion, in a second step said cellular components are stained with antibodies to the progenitor cell markers, and in a third step the progenitor cells are separated from the other cells directly or indirectly by means of the attached antibodies, preferentially, but not WO 2005/061696 PCT/CH2004/000738 4 exclusively, by using the above-mentioned magnetic beads. To this end, the antibody-stained progenitor cells are attached to beads, preferably small iron beads, and the progenitor cells are extracted by means of the beads, preferably in case of small iron beads by using a magnet, and wherein subsequently the beads as well as if need be the antibodies are removed from the progenitor cells. This is for example possible by selecting the beads, which have been provided with specific antibodies attached to the beads, which antibodies selectively bind to the progenitor cells. To obtain the pure cells, subsequently the beads are removed from the progenitor cells, which is for example possible by means of enzymes cleaving the link between the beads and the antibodies. If the link berween the beads and the antibodies is based on DNA, such cleavage can be effected by using Dnase, in case where the link between the beads and the antibodies is based on amino acid chains, protcinases can be used. Surprisingly, while normally progenitor cells have to be cultured , i.e. grown, on very specific feeder layers, like for example mouse fibroblast feeder layers, the progenitor cells isolated in the present method do not need such specific feeder layers, but can generally be grown on other feeder layers based on for example the ones disclosed within the scope of the specific examples. More specifically, the method of isolation comprises the following steps: (i) the whole human mammary secretion is subjected to centrifugation generally leaving a fat layer on top, a protein and carbohydrate rich supernatant beneath it, and at the bottom a pellet of cells; (ii) fat fraction and supernatant are removed; (iii) e.g. a buffer, such as, but not limited to, phosphate buffered saline, tris buffer saline, TBS and/or PBS, or media, such as, but not limited to, Williams media or RPMI Media, is added and the cells (not only comprising progenitor cells) arc resuspended in the buffer / media and centrifuged as before, preferentially repeating this process 3 or 4 times, leaving a substantially pure cell pellet; (iv) the progenitor cells are separated from the cell pellet. Preferentially, the separation of the progenitor cells from the cell pellet is carried out in the following steps:(v) the cell pellet is suspended in media, preferentially in RPMI media containing for example fetal calf (bovine) scrum; (vi) this suspension is incubated WO 2005/061696 PCT/CH20O4/0OO738 5 with (magnetic) beads which have preferentially before been incubated with progenitor-specific antibodies (preferentially stem cell-specific antibodies, like anti-mouse IgG antibodies), which antibodies are attached to the magnetic beads via a small strand for example of DNA or ami no acids, wherein the incubation of the cell suspension in these magnetic beads is preferentially carried our for 15 minutes at 4°C; (vii) once the progenitor cells have bound to the magnetic beads a magnet is attached to the tube containing the cells/beads, thus attracting the progenitor cells connected with the beads to the magnet, whereas unbound cells are not and remain in the supernatant; (viii) removing the supernatant leaving only the progenitor cells bound to the beads via the progenitor cell antibody. It has to be pointed out that other types of beads or generally separation means can be used which allow to selectively attach them to the progenitor cells. Such beads have to be separated from the progenitor cells, and to this end preferentially the following processing steps can be used: (ix) progenitor cells bound to the beads via the stem cell antibody are removed by an appropriate cleavage means, preferentially, in case of the antibody being attached to the beads via small strand of DNA, a by means of addition of a Dnase, (x) the beads are removed by attaching the magnet once more, such that the beads, no longer attached to the stem cells, are attracted to it; (xi) removing the supernatant now containing the isolated progenitor cells. An alternative method of separation which is based on spcific growth of pluripotent cells involves the following steps: (i) a first step the whole human mammary secretion is subjected to centrifugation leaving a fat layer on top, a protein and carbohydrate rich supernatant beneath it, and at the bottom a pellet of cells. (ii) in an optional second step, the cell pellet is washed in media. Preferably the cells are washed in RPMI media only. /in) in a third step the cells of the cell pellet are plated onto a cell culture treated device in growth media and arc allowed to incubate. Preferably this incubation is carried out for 10-30 days, most preferably for 14-20 days. WO 2005/061696 PCT/CH2OO4/OOO7J8 6 (iv) the cells axe harvested, i.e. they are detached from the support, preferably by trypsination. Subsequently the detached cells are washed, preferably using growth media solution. (v) the harvested and washed cells are plated onto a reconstituted basement membrane preparation for growth, preferably up to confluence. In this last step, preferably a solubulizcd basement membrane preparation extracted from EHS mouse sarcoma is used, like e.g. Matrigel™ as available from BD Bioscienccs. This method leads to cell cultures with a morphology which is typical for progenitor cells. The cells do not look like lactocytes and due to the growth media which is permissive for progenitor/stem cell/lactocyte growth, they cannot be bacterial cells. The present invention furthermore relates to progenitor cells, which are preferentially pluripotent or multipotent progenitor (stem) cells, derived using a method as described above. Additionally, the present invention relates to uses of such progenitor cells, for example for ex vivo, in vitro and/or in vivo applications. Without limiting the scope of the invention, such use may extend to the following specific examples or combinations thereof : creation of tissues or cells for the benefit of the mother and/or of the infant and/or of other individuals; subsequent gene therapy treatments or intrauterinc foetal treatments; generation of cells, tissue, glands or organs for the treatment of disease; subsequent cloning or scientific research; one or several of the group of the following purposes: clinical, diagnostic, bioengineering, lactoengineering, breast tissue regeneration, breast reconstructive surgery, breast cosmetic or enhancement surgery, exocrine gland tissue regeneration and/or surgery. Further embodiments of the present invention are outlined in the dependent claims. SHORT DESCRIPTION OF THE FIGURES In the accompanying drawings preferred embodiments of the invention are shown in WO 2005/061696 PCT/CH2OO4/OOO738 7 which: Figure I shows the total mixed cell population of human milk following removal of the fat and skim milk; Figure 2 shows the stem cells (sc) following their isolation from a total milk cell population. These cells have been cytospun onto microscope slides and stained with hacmatoxylin and eosin. Figure 2a) and 2b) show a single stem cell (sc). Figure 2c) shows a single stem cell still bound to the Dynabcads (Db). The Dynabeads* (Db) are 4.5nM in size, thereby permitting the approximate sizing of the stem cells to be 6-7^M; Figure 3 following purification of the stem cells from a total milk cell population, the stem cells that are isolated can be cultured using a variety of culture conditions; this figure shows a single stem cell 1 day after being placed into culture; Figure 4 after 1 to 2 weeks in culture, the stem cells isolated from a total cell population begin to undergo cell division (mitosis); the arrow in this photo indicates a cell undergoing mitosis; Figure 5 after several months in culture, these cells do not appear to have differentiated into other cell types; this figure shows a stem cell after 2 months of culture which has been cytospun onto a microscope slide and stained with haematoxylin and eosin; Figure 6 after 2 months in culture the stem cells have not differentiated; this figure shows the stem cells isolated with the SSEA-4 (a) and Tra 1-60 (b) antibodies, following 2 months in culture; the cells were washed out of the culture medium and cytospun onto a microscope slide before being visualised on a confocal microscope with the SSEA-4 (a) and Tra 1-60 (b) antibodies conjugated to a fluorescently labelled secondary antibody (goat anti-mouse IgG conjugated to AlexaFluor-488). Figure 7 shows the growth rate of isolated stem cells in culture; cells were seen to WO 2005/061696 PCT/CH20O4/OO0738 8 divide and proliferate over a period of 4 days; cells isolated with Dynabeads* and antibody SSEA-4 have shown an increase in total number of cells from approximately 50 cells per plate to 150 cells pcT plate in 4 days of culture; the central area of the plates was determined and a cruciform counting pattern was used; N=10 fields in each culture and numbers on individual days were simply totalled; the data shown in this Figure is the same experiment performed in triplicate. Figure 8 shows a monolaycr of pluripotcnt cells as obtained from mammalian secretion using an alternative procedure according to the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS To determine whether a cell believed to be a stem cell is actually a stem cell, it has historically been necessary to undertake transplantation of the cells of interest into sub-lethally irradiated mice. If these transplanted cells subsequently locate and repopulate any organs of these mice, the cells in question have been considered to be pluripotent stem cells. However, more recently the identification of extracellular markers for pluripotcnt stem cells (for example Tra-1-60 and SSEA-4; Chcmicon International, Temccula, California, US) have enabled the identification of pluripotent (stem) cells without the prolonged process of transplantation. After washing all cells out of human milk by a repeating the process of gentle centrifugation, aspiration of the supernatant and the rcsupension of the cells in a buffer or media several times, the stem cells were isolated from a total milk cell population (Figure 1) using dynabeads according to manufacturers specifications. As buffer Tris-buffcTed saline solutions (TBS) or phosphate buffered saline solutions (PBS) can be used. In particular, TBS-soIutions which are 10 mM in Tris, 150 mM in NaOH and which arc adjusted to have a pH around 7.4 can be used. In case of PBS-solutions, those maybe 1.1 mM KH2PO4, 140mM NaCl, 4.5mM NajHPO4.7H2O and 2.7mM NaCI, and may be adjusted to have a pH around 7.4. As media, Williams or RPMI (Roswell Park Memorial Institute) as available from Sigma Aldrich, US, under the product numbers WO 2005/06)696 PCT/CH 2004/0007J8 9 R6504, R7755, R4130 or W4125, W4128, W1878, can be used. Also possible as media is RPMI from Gibco (CA, US) Cat. No 11875-093. Stem cell-specific antibodies, e.g. Tra-1-60, Tra 1-81 and/or SSEA-4, both monoclonal, both e.g. available from Chemicon international, CHEMICON International, 28820 Single Oak Drive, Temecula CA 92590, were attached to the Dynabcads* for isolation of the progenitor cells. Also possible is the use of haematopoietic cells antibody CD 133 (cat* MAB1133) from R&D Systems, Inc CA, US. Primitive haematopoietic stem and progenitor cells from peripheral blood have already been isolated using the immuno-magnctic cell separation principles. Research groups using monoclonal-antibody conjugated magnetic particles (CliniMACS System and Reagent AC133 from Miltcnyi Biotcc) have successfully isolated and cultured CD133 positive cells. Any stem cell-specific antibody (inclusive of; SSEA-3, SSEA-1 and TRA 1-81 Oct-4, CD 133) are incubated with Dynabeads". Dynabeads* are available from Dynal AS, NO, and are small iron beads, that have an anti-mouse igG antibody attached to it via a small strand of DNA. Possible are for example Dynabcads* available under the name Dynal CD34. Incubation of the Dynabcads* is carried out for lh at room temperature prior to the Dynabeads being incubated with the cells isolated from milk for 15 minutes at 4°C. Once the stem cells have bound to the Dynabcads which typically takes about 30 minutes to 1 hour, a magnet is attached to the side of the tube containing the cells/Dynabeads. Dynabeads are uniform, polystyrene-based, paramagnetic, beads with 4.5 urn in diameter. The Dynabeads' (with the stem cells attached) are attracted to the magnet, whereas the unbound cells arc not and remain in the supernatant. The supernatant is then removed leaving only the cells bound to the Dynabcads via the stem cell antibody. The cells bound to the Dynabcads* via the stem cell antibody arc removed by the addition of Dnase breaking the small strand of DNA. This is called releasing buffer and is part of the DynaJ kit 62500 U/ml (15000-20000U per vial quoted in the instructions). The Dynabeads* are removed by attaching the magnet once more to which the Dynabeads, no longer attached to the stem cells, arc attracted. The WO 2005/061696 PCT/CH 2004/000738 10 supernatant now contains the stem cells which arc removed. These stem cells can now be used for any subsequent application as listed above/below. The isolated progenitor (stem) cells can be cultured on mouse embryo fibroblast feeder cells in Knockout-Dulbccco's modified Eagle's medium. Typically cultivation can be carried out at a temperature of 37°C. Examples of the use of feeder cells: • Prolonged propagation of human embryonic stem cells is cunently achieved by coculture with primary mouse embryonic fibroblasts serving as feeder cells. • Acceleration of the formation of cultured epithelium using the sonic hedgehog expressing feeder cells. • Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture. • Proliferation and maturation of embryonic stem cells into endothelial cells in an in vitro model of vasculogenesis required OP9 feeder cells. • Selective expansion and continuous culture of macrophages from adult pig blood. Macrophages were selectively expanded and continuously cultured from adult pig blood directly into the medium overlaying a feeder layer of STO mouse fibroblasts. • Establishment and characterization of a novel human immature megakaryoblastic leukemia cell line, M-MOK, dependent on fibroblasts for its viability. A novel fibroblast-dependent human immature megakaryoblastic leukemia cell line (M-MOK) was established from the bone marrow of a girl with acute megakaryoblastic leukemia, and its growth was determined to be completely dependent on the presence of human embryonic lung-derived fibroblasts, HEL-O. • In vitro culture of embryonic disc cells from porcine blastocysts using foetal G30 porcine fibroblasts which had been previously irradiated as a feeder layer. WO 2005/061696 PCT7CH2004/0OO738 11 • Acute lyinphocytic leukemias (ALL) cells of infants and children were found to preferentially survive in coculture with a cloned cell line of cndothelial adipose cells (14F1.1) from mouse bone marrow and exhibited extensive growth in the presence of the mouse stromal cells. These ALL cells were strictly dependent upon the mouse stromal clone 14F1.I and failed to proliferate in the absence of the endothelial adipocytcs or with a variety of feeder cells. Cell growth Cells isolated with Dynabcads and antibody SSEA-4 have shown an increase in total number of cells from approximately 50 cells per plate to 150 cells per plate in 4 days (sec Figure 7). Methods: Cell preparation 150 ml of whole milk is spun for 15 minutes at 2000rpm to collect cells. Cell pellet is resuspended in approximately 4ml TBS 1% BSA and ccnlrifuged again for 10 minutes. Final pellet is resuspended in 200^1 of RPMI 1% FCS. Bead preparation Beads are washed 3x in RPMI 1% foetal calf serum. Beads coated with primary antibody at a concentration of lul in 500 u! TBS 1% BSA and incubated for 1 hour at room temperature with gentle mixing. Coated beads separated and washed 3x in lml TBS 1% BSA and transferred to clean tube. Final wash in lml RPMI 1% FCS. Cell isolation 200 \i\ cell preparation added to beads and incubated for 30min at approximately 100°C. WO2005/U616V6 PCT/CH 2004/000738 12 Following incubation the unbound fraction is discarded. 200 ul RPMI 1% FCS is added to the cell and bead complex which is then gently pipetted to release further unbound matter. To 200 ul of bead and cell complex add 5 ul releasing buffer and incubate for 15 minutes at 37°C with gentle mixing. Vigorously pipette bead plus cell complex to help release cells. Collect the 200 ul of isolated cell suspension and add to clean Eppcndorf. This cell suspension can then be immediately introduced into the culture system. Culture Method Cells were seeded on collagen-coated culture plates. Following a settling period the supernatant was carefully drawn off to remove contaminants and fresh media added. Cultures were then incubated in plates at 37°C in a 5% COj incubator with medium changes every two days. Culture media William's E medium supplemented with 10% fetal calf serum; 10'7M dexamethasone (Sigma); Gluiamine 2mmol/L ITS+ prcmix containing insulin (6.25ug/ml), transferrin (6.25ug/ml), sclcnious acid (6.25ng/ml), bovine serum albumin (1.25mg/ml) and linoleic acid (5.35yg/ml) from Becton-Dickinson, Bedford, MA. Penicillin / streptomycin 5,000 ng/ml Fungizone (25Oug/ml) Antibodies An example of some of the potential antibodies that can be used in this system arc; (All supplied by CHEMICON International, 28820 Single Oak Drive, Temccula CA 92590) SSEA-I (Cat number MAB4301) WO 2005/061696 PCT/CH2004/000738 13 SSEA-3 (MAB4303) SSEA-4 (MAB4304) TRA 1-60 (MAB4360) TRA 1-81 (MAB4381) haematopoietic cells antibody CD 133 (cattf MAB1133) from R&D Systems, Inc. Buffers TBS 1% BSA, lOmM Tris, 150 mM NaOH RPMI 1%FCS PBS 1.1 mM KH2PO Following purification of the stem cells from human milk, using the extracellular markers for stem cells (Tra-1-60 and SSEA-4) bound to Dynabeads* we cytospun the stem cells onto microscope slides and stained the stem cells using haematoxylin and eosin (Figure 2). Using these same purified stem cells we have been able to culture the cells (Figure 3) and demonstrate that these cells do undergo cell division (Figure 4). Following prolonged culture, we have been able to stain these cells using haematoxylin and cosin (Figure 5), and have demonstrated that these cells remain stem cells as they continue to bind the stem cell antibodies (Tra-1-60 and SSEA-4) following several months of culture, as visualised by confocal microscopy (Figure 6). Growth of the isolated cells was also verified, see Figure 7. An alternative way of isolation/growth of the pluripotent cells is using a specific combination of growth media by means of which it is possible to grow a monolayer of pluripotent cells that clearly do not look like lactocytes (see Fig. 8). Indeed the grown cells in culture are physically identical to for example liver stem cell cultures. Further WO 2005/061696 PCT/CH20O4/00O738 14 classification of these cells by the cellular markers they express and by their gene activity is possible. This alternative way of isolation or selective growth can be carried out as follows: Preparation of Matrigel™ coated plates: The Matrigel (commercially available reconstituted basement membrane, BD Matrigel™, BD Bioscicnccs rcf-354234) was allowed to defrost slowly on ice. Pipette tips and culture plates were cooled and kept on ice prior to use. 50ul of the thawed Matrigel was aliqouted and spread with a pipette tip into each of the wells of a 24 well flat bottom microplatc and allowed to incubate on ice for 30 minutes. Following this the plates were further incubated for 30 minutes at 37 degrees centigrade in order to set the gel. Once set lml of growth media was aliquoted to each well and the plate returned to the incubator until required (no longer than 5 days). Growth and preparation of cells Freshly expressed hind milk (approximately 50ml, hind milk is human breastmilk that is high in fat content and creamy in color, it provides the bulk of the baby's calories and is most responsible for weight gain) was centriftjged to pellet the cells (details as given above). The cell pellet was washed 2x in RPM1 media (RPMI Medium 1640, Gibco ref-108-36) only. Cells were then plated onto cell culture treated plastic culture dishes in growth media (Cell culture growth media composition: RPMI 1640; Fetal Bovine scrum 20*/»; Insulin 5ug/ml; Cholera toxin 50ng/ml; Hydrocortisonc 0.5ug/ml; Penicilin streptomycin fungjzone 2x) and allowed to incubate for approximately 14-20 days. After this period plates showing large colonies and approaching confluence were selected and the cells harvested by trypsinization (Trypsin-EDTA lx, JRH Bioscicnce WO 2005/061696 PCT/CH2O04/00O738 15 ref=59218). The harvested cells were washed Ix in growth media and plated onto Matngel™ coated plates at a density approximately 1500 cells/ml. After around 14 days patches of confluent cells can be observed. Description The morphology is typically epithelial in that confluent sheets of cells develop and the cells within the colonies are closely associated. The fact that breastmilk derived cells cultured in this manner will proliferate in primary culture and form extensive circular colonies with well defined borders is strongly indicative of cells with progenitor like qualities. That the cells appear undiffercntiated and many can be observed to have a large nucleus to cytoplasm ratio is even more evidence for a progenitor like identity. Specific materials used Cholera toxin (LIST BIOLOGICAL LABORATORIES ref-lOlB) Fetal Bovine serum (Invitrogen rcf-10099141) Hydrocortisone (Sigma ref-HO135) Insulin (Sigma ref-19278) Pcncillin/Strepomycin Fungizone (Scott Scientific ref-17.745E) MICROPLATE (IWAKJ rcf- 3820-024) Cell Culture Dish (Coming ref-430165) Due to the plasticity of the pluripotent stem cells, these isolated cells can be utilised for a multitude of different applications. For example these cells can be; • used to create tissues for the benefit of the mother and infant (and potentially WO 20OS/W1696 PCT/CH 2004/000738 16 other individuals), including gene therapy treatments, intrauterine foetal treatments, and the generation of cells, tissue, glands, or organs for the treatment of disease. This includes their use in scientific research, clinical, diagnostic or commercial applications. This can also include the generation of biological compounds including cells, cellular compartments, cellular secretions, cell isolates, nucleotides, deoxyribonucleic acids, amino acids, proteins, glycoproteins, carbohydrates, lipids, hormones, growth factors, and cytokines. Furthermore, this can include the generation of cells as a precursor, or as a consequence, of the generation of tissue, glands or organs for the treatment of disease, tissue regeneration, body enhancement, or cosmetic applications for the following tissues; Olfactory, Auditory, Optical, Lymphatic, Immune, Haematopoietic, Endocrine, Exocrine, Bowel, Gastrointestinal, Pcycrs Patches, Islets of Langerhans, Skeletal, Muscle, Connective, Vascular, Blood, Skin, Hair, Nails, Mammary, Brain and Central Nervous System, Liver, Heart, Lung, Kidney, Bone, Pancreas, Reproductive. • stored for future. The subsequent use of these stem cells, or cells differentiated or differentiated could include storage of these stem cells for future use as outlined below. This includes their storage for use in scientific research, clinical, diagnostic or commercial applications. • used for cell culture, whether this be for propagation of these same stem cells, or for the differentiation or dedifferentiation of these stem cells into another cell type. This includes their use in scientific research, clinical, diagnostic or commercial applications. • used for cloning. The subsequent use of these stem cells, or cells differentiated or dedifferentiatcd from these stem cells could be used to generate clones, whether embryonic or whole animal. This includes their use in scientific research, clinical, diagnostic or commercial applications. • used for scientific research. The subsequent use of these stem cells, or cells differentiated or dediffcrentiated from these stem cells could be used in scientific WO 200.V061696 PCT/CH20O4/UO0738 17 research. This can include the generation of biological compounds including cells, cellular compartments, cellular secretions, cell isolates, nuclcotides, dcoxyribonucleic acids, ami no acids, proteins, glycoproteins, carbohydrates, lipids, hormones, growth factors, and cytokincs. In addition this could include the generation of cells as a precursor, or as a consequence, of the generation of tissue, glands or organs for the treatment of disease, tissue regeneration, body enhancement, or cosmetic applications for the following tissues; Olfactory, Auditory, Optical, Lymphatic, Immune, Haematopoietic, Endocrine, Exocrine, Bowel, Gastrointestinal, Peycrs Patches, Islets of Langerhans, Skeletal, Muscle, Connective, Vascular, Blood, Skin, Hair, Nails, Mammary, Brain and Central Nervous System, Liver, Heart, Lung, Kidney, Bone, Pancreas, Reproductive. • used for clinical, diagnostic or commercial applications. The subsequent use of these stem cells, or cells differentiated or dedifferentiated from these stem c«lls could be used in clinical, diagnostic or commercial applications. This can include the generation of biological compounds including cells, cellular compartments, cellular secretions, cell isolates, nuclcotides, deoxyribonucleic acids, amino acids, proteins, glycoproteins, carbohydrates, lipids, hormones, growth factors, and cytokines. In addition this could include the generation of cells as a precursor, or as a consequence, of the generation of tissue, glands or organs for the treatment of disease, tissue regeneration, body enhancement, or cosmetic applications for the following tissues; Olfactory, Auditory, Optical, Lymphatic, Immune, Haematopoietic, Endocrine, Exocrine, Bowel, Gastrointeitinal, Pcyers Patches, Islets of Langerhans, Skeletal, Muscle, Connective, Vascular, Blood, Skin, Hair, Nails, Mammary, Brain and Central Nervous System, Liver, Heart, Lung, Kidney, Bone, Pancreas, Reproductive. • used for bioengineering. The subsequent use of these stem cells, or cells differentiated or dediffereniiatcd from these stem cells could be used to generate any other cell type in the human body. These cells, tissues, or organs could then be used for cosmetic/reconstructive surgery, organ/tissue transplantation or the WO 200W061696 PCT/CH20O4/000738 18 generation of cells/tissue/organs for a third party. This can include the generation of biological compounds including cells, cellular compartments, cellular secretions, cell isolates, nucleotides, deoxyribonuclcic acids, amino acids, proteins, glycoproteins, carbohydrates, lipids, hormones, growth factors, and cytokines. In addition this could include the generation of cells as a precursor, or as a consequence, of the generation of tissue, glands or organs for the treatment of disease, tissue regeneration, body enhancement, or cosmetic applications for the following tissues; Olfactory, Auditory, Optical, Lymphatic, Immune, Haematopoietic, Endocrine, Exocrinc, Bowel, Gastrointestinal, Peycrs Patches, Islets of Langcrhans, Skeletal, Muscle, Connective, Vascular, Blood, Skin, Hair, Nails, Mammary, Brain and Central Nervous System, Liver, Heart, Lung, Kidney, Bone, Pancreas, Reproductive. • used for lactoengineering. The subsequent use of these stem cells, or cells differentiated or dedifferentiated from these stem cells could be used to generate biological compounds of milk including cells, cellular compartments, cellular secretions, cell isolates, nucleotides, deoxyribonucleic acids, amino acids, proteins, glycoproteins, carbohydrates, lipids, hormones, growth factors, and cytokines. • breast tissue regeneration. The subsequent use of these stem cells, or cells differentiated or dedifferentiated from these stem cells could be used to generate breast tissue. • breast reconstructive surgery. This regenerated tissue as above could then be used for reconstructive breast surgery. • breast cosmetic surgery This regenerated tissue as above could then be used for cosmetic breast surgery. • exocrinc gland tissue regeneration and/or surgery. The subsequent use of these stem cells, or cells differentiated or dedifferentiated from these stem cells could be used to generate exocrine gland tissue, which in rum, could be used for the regeneration or replacement of exocrine glands. WO 2005/06)696 PCT/CH2004/000738 19 the generation of biological compounds including cells, cellular compartments, cellular secretions, cell isolates, nuclcotides, dcoxyribonucleic acids, amino acids, proteins, glycoproteins, carbohydrates, lipids, hormones, growth factors, and cytokines. WO 2005/061696 PCT/CH20O4/O0O7J8 20 CLAIMS 1. A method for isolating progenitor cells from a human body, inclusive of all cells with stem cell-like characteristics, wherein such cells are directly or indirectly derived from human mammary secretion, be it colostrum, mature milk, or dry period secretion from males or females, of said human body during at least one of the following periods: non-pregnant period, pregnant period, lactating period, involuting period. 2. A method according to claim 1, wherein the progenitor cells are pluripotcnt or multipotent. 3. A method according to claim I, wherein said progenitor cells arc isolated from the mammary secretion in that noncellulax parts of the mammary secretion arc separated from the cellular parts. 4. A method according to claim 3, wherein non-pluripotent or non-multipotcnt cells arc removed from cellular parts. 5. A method according to any of the preceding claims, wherein secretory epithelial cells, leucocytes and in particular nonhuman cells like bacterial cells are removed from the mammary secretion. 6. A method according to any of the preceding claims, wherein the mammary secretion during lactating periods is used for the isolation of the progenitor cells, and wherein the mammary secretion during particular stages of mammary secretion such as: after beginning of individual feeding; versus end of individual feeding; lactation phase; preferably early lactation. WO 2005/061696 PCT/CH20O4/0OO738 21 7. A method according to any of the preceding claims, wherein magnet beads are used to ihc isolation of the progenitor cells. 8. A method according to any of the preceding claims, wherein in a first step cellular components axe washed out of the mammary secretion, in a second step said cellular components are stained with antibodies to the progenitor cell markers, and in a third step the progenitor cells arc separated from the other cells directly or indirectly by means of the attached antibodies. 9. A method according to claim 8, wherein the antibody-stained progenitor cells are attached to beads, preferably small iron beads, and wherein the progenitor cells are extracted by means of the beads, preferably in case of small iron beads by using a magnet, and wherein subsequently the beads as well as if need be the antibodies are removed from the progenitor cells. 10. A method according to claim 9, wherein removal of the beads is effected by means of enzymes selected from the following group: Dnase, Proteinase, Rnase. 11. A method according to any of the preceding claims, wherein the progenitor cells are cultured without using a fibroblast feeder layer, in particular without using a mouse fibroblast feeder layer. 12. A method according to any of the preceding claims, wherein in (i) a first step the whole human mammary secretion is subjected to centrifugation leaving a fat layer on top, a protein and carbohydrate rich supernatant beneath it, and at the bottom a pellet of cells; WO 2005/061696 PCT/CH20O4/OOO738 22 (ii) in a second step fat fraction and supernatant are removed; (iii) in a third step a buffer, such as, but not limited to, phosphate buffered saline, tris buffer saline, TBS and/or PBS, or media, such as, but not limited to, Williams media or RPMI Media, is added and the cells are rcsuspended in the buffer / media and centrifuged as before, preferentially repeating this process 3 or 4 times, leaving a substantially pure cell pellet; (iv) and in a fourth step the progenitor cells are separated from the cell pellet. 13. A method according to any preceding claim, wherein a oell pellet is generated from the human mammary secretion, and subsequently the following separation steps are used: (v) the cell pellet is suspended in media, preferentially in RPMI media containing foetal calf (bovine) serum, (vi) this suspension is incubated with magnetic beads which have before been incubated with progenitor, preferentially stem cell-specific antibodies, like anti-mouse IgG antibodies, which antibodies arc attached to the magnetic beads via a small strand of DNA, wherein the incubation of the cell suspension is preferentially carried our for 15 minutes at 4°C; (vii) once the progenitor cells have bound to the magnetic beads a magnet is attached to the tube containing the cells/beads, thus attracting the progenitor cells connected with the beads to the magnet, whereas unbound cells are not and remain in the supernatant; (viii) removing the supernatant leaving only the progenitor cells bound to the beads via the progenitor cell antibody. 14. A method according to claim 13, wherein subsequently, the following steps are used: (ix) progenitor cells bound to the beads via the stem cell antibody arc removed by an appropriate cleavage means, preferentially, in case of the antibody being WO 2005/061696 PCT/CH20O4/0O0738 23 attached to the beads via small strand of DNA, a by means of addition of a Dnase, (x) the beads arc removed by attaching the magnet once more such that the beads, no longer attached to the stem cells, are attracted to it; (xi) removing the supernatant now containing the isolated progenitor cells. 15. A method according to any of claims 1-12, wherein the cells are separated from human mammary secretion by centrifugation, subsequently incubated in a growth media that is permissive for progenitor/stem cell/lactocyte growth. 16. A method according to claim 15, wherein in (i) a first step the whole human mammary secretion is subjected to centrifugation leaving a fat layer on top, a protein and carbohydrate rich supernatant beneath it, and at the bottom a pellet of cells; (ii) in a second step, the cell pellet is washed in media, preferably in RPMI media only (iii) in a third step the cells of the cell pellet are plated onto a cell culture treated device in bacteriocidal and/or fungicidal growth media and are allowed to incubate, preferably for 10-30 days, most preferably for 14-20 days, (iv) the cells are harvested, preferably by trypsination, and washed, preferably using growth media (v) the harvested cells are plated onto a reconstituted basement membrane preparation for growth preferably up to confluence. 17. A method according to claim 16, wherein in step (v) a solubulized basement membrane preparation extracted from EHS mouse sarcoma is used, as e.g. Matrigel™. WO 2005/061696 PCT/CH 2004/000 7 J8 24 18. Progenitor cells, preferentially pluripotent or multipoint progenitor cells, derived using a method according to any of the preceding claims 1 through 17. 19. Use of pluripotent or multipotcnt progenitor cells as derived using a method according to any of the claims 1-17 for ex vivo, in vitro and/or in vivo applications. 20. Use according to claim 19, to create tissues or cells for the benefit of the mother and/or of the infant and/or of other individuals. 21. Use according to claim 17 or 20, including subsequent gene therapy treatments or intrauterine foetal treatments. 15 22. Use according to claim 19-21, for the generation of cells, tissue, glands or organs for the treatment of disease. 23. Use according to any of the claims 19-23, for subsequent cloning or scientific research. 24. Use according to any of the claims 19-23, for one or several of the group of the following purposes: clinical, diagnostic, bioengineering, lactoengincering, breast tissue regeneration, breast reconstructive surgery, breast cosmetic or enhancement surgery, exocrine gland tissue regeneration and/or surgery.

Full Text

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SPECIFICATION
TITLE Method for isolating cells from mammary secretion
TECHNICAL FIELD
The present invention relates to a method for isolating cells from a human body, as well as uses of such cells.
BACKGROUND OF THE INVENTION
Stem cells are defined as clonogenic, self-renewing progenitor cells that can generate a wide variety of more specialised cell types via the process of differentiation. Classically, it has been believed that there are two distinct types of stem cell. Embryonic stem (ES) cells are derived from the inner mass of the blastocyst, are pluripotent and thus are capable of generating into all differentiated cell types within the body. The other sub-population of stem cells are derived from ES cells and arc organ-or tissue-specific. These multipotcnt cells, also known as adult stem cells, were believed to be able to differentiate only into tissues from their organ of origin. An example of these multipotcnt cells are haematopoietic stem cells, which serve to continually regenerate the cells of the blood and immune system.
Stem cells have been isolated from a wide range of tissues, from those that have a high rate of ongoing cellular turnover, such as blood, cord blood, bone marrow, skin, intestine, and breast tissue, to those with a low turnover such as brain, skeletal muscle.

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and juvenile teeth. Irrespective of the tissue of origin, a long standing dogma has been that adult stem cells can only differentiate into the tissues from which they were derived. However recent work has demonstrated that upon exposure to a novel environment, organ-specific stem cells can overcome these intrinsic restrictions to transdifferentiatc into other tissues. For example it has been shown that neural stem cells can transdiffercntiate into blood cells, bone-manow derived stem cells can transdifferentiate into muscle, brain, liver and heart cells, and skin derived stem cells can transdifferentiate into brain cells. Therefore it now appears likely that the dogma associated with developmental restriction of organ-specific stem cells is incorrect and it is feasible that these ES cells under appropriate environmental stimuli, can transdifferentiatc into another cell type.
Human milk contains a mixture of different cell types. Secretory epithelial cells (lactocytes) are found in milk due to them sloughing off the basement membrane of the breast as a consequence of the pressure associated with the continued filling and emptying of the breast. Lactocytes account for approximately 10-20% of the total cell population. The majority of the remainder of cells found in human milk are leukocytes (immune cells such as lymphocytes, macrophages, monocytes, natural killer cells, basophils, cosinophils, and neutrophils), and are believed to be in milk to both protect the breast from infection and to provide immune protection for the infant. To date, these arc the only cell types believed to be contained in milk.
SUMMARY OF THE INVENTION
The objective problem underlying the present invention is therefore to provide a new method for isolating progenitor cells from the human body. In this context, the term progenitor cells shall include all cells with stem cell-like characteristics, preferentially but not exclusively including pluripotent or multipotent progenitor cells like for example stem cells.
The present invention solves the above problem by deriving such cells directly or indirectly from human mammary secretion, be it colostrum, mature milk, or dry period

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secretion from males or females, of said human body during at least one of the following periods: non-pregnant period, pregnant period, lactating period, involuting period. In other words, here we demonstrate that surprisingly, progenitor cells can also be found in human lactation milk and that these cells have the potential to be utilised for the generation of tissues for the mother and infant. It has to be noted that not only human mammary secretion but generally mammary secretion from mammalian species can be used for the isolation of corresponding progenitor cells. It can unambiguously be shown by means of progenitor-cell-specific antibodies, that indeed mammalian secretion, i.e. for example human milk, comprises progenitor cells.
In a first preferred embodiment of the present invention, said progenitor cells are isolated from the mammary secretion in that non-cellular parts of the mammary secretion are separated from the cellular parts and that in particular non-pluripotcnt or non-multipotent cells are removed from cellular parts thus derived. The cellular parts of mammary secretion, apart from pluripotent cells may further comprise secretory epithelial cells, leucocytes and in particular nonhuman cells like bacterial cells. Those non-pluripotent cells are preferentially removed from the mammary secretion.
According to another preferred embodiment of the present invention, the mammary secretion during lactating periods is used for the isolation of the progenitor cells, wherein the mammary secretion during particular stages of mammary secretion such as: after beginning of individual feeding; versus end of individual feeding; lactation phase; preferably early lactation, is used.
A particularly useful and practicable way of isolating those progenitor cells from the secretion is possible if magnet beads are used. Those magnetic beads are to this end preferentially connected to progenitor-cell-specific antibodies allowing the attachment of the beads to the progenitor cells.
Typically, in a first step cellular components are washed out of the mammary secretion, in a second step said cellular components are stained with antibodies to the progenitor cell markers, and in a third step the progenitor cells are separated from the other cells directly or indirectly by means of the attached antibodies, preferentially, but not

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exclusively, by using the above-mentioned magnetic beads. To this end, the antibody-stained progenitor cells are attached to beads, preferably small iron beads, and the progenitor cells are extracted by means of the beads, preferably in case of small iron beads by using a magnet, and wherein subsequently the beads as well as if need be the antibodies are removed from the progenitor cells. This is for example possible by selecting the beads, which have been provided with specific antibodies attached to the beads, which antibodies selectively bind to the progenitor cells. To obtain the pure cells, subsequently the beads are removed from the progenitor cells, which is for example possible by means of enzymes cleaving the link between the beads and the antibodies. If the link berween the beads and the antibodies is based on DNA, such cleavage can be effected by using Dnase, in case where the link between the beads and the antibodies is based on amino acid chains, protcinases can be used.
Surprisingly, while normally progenitor cells have to be cultured , i.e. grown, on very specific feeder layers, like for example mouse fibroblast feeder layers, the progenitor cells isolated in the present method do not need such specific feeder layers, but can generally be grown on other feeder layers based on for example the ones disclosed within the scope of the specific examples.
More specifically, the method of isolation comprises the following steps: (i) the whole human mammary secretion is subjected to centrifugation generally leaving a fat layer on top, a protein and carbohydrate rich supernatant beneath it, and at the bottom a pellet of cells; (ii) fat fraction and supernatant are removed; (iii) e.g. a buffer, such as, but not limited to, phosphate buffered saline, tris buffer saline, TBS and/or PBS, or media, such as, but not limited to, Williams media or RPMI Media, is added and the cells (not only comprising progenitor cells) arc resuspended in the buffer / media and centrifuged as before, preferentially repeating this process 3 or 4 times, leaving a substantially pure cell pellet; (iv) the progenitor cells are separated from the cell pellet.
Preferentially, the separation of the progenitor cells from the cell pellet is carried out in the following steps:(v) the cell pellet is suspended in media, preferentially in RPMI media containing for example fetal calf (bovine) scrum; (vi) this suspension is incubated

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with (magnetic) beads which have preferentially before been incubated with progenitor-specific antibodies (preferentially stem cell-specific antibodies, like anti-mouse IgG antibodies), which antibodies are attached to the magnetic beads via a small strand for example of DNA or ami no acids, wherein the incubation of the cell suspension in these magnetic beads is preferentially carried our for 15 minutes at 4°C; (vii) once the progenitor cells have bound to the magnetic beads a magnet is attached to the tube containing the cells/beads, thus attracting the progenitor cells connected with the beads to the magnet, whereas unbound cells are not and remain in the supernatant; (viii) removing the supernatant leaving only the progenitor cells bound to the beads via the progenitor cell antibody.
It has to be pointed out that other types of beads or generally separation means can be used which allow to selectively attach them to the progenitor cells. Such beads have to be separated from the progenitor cells, and to this end preferentially the following processing steps can be used: (ix) progenitor cells bound to the beads via the stem cell antibody are removed by an appropriate cleavage means, preferentially, in case of the antibody being attached to the beads via small strand of DNA, a by means of addition of a Dnase, (x) the beads are removed by attaching the magnet once more, such that the beads, no longer attached to the stem cells, are attracted to it; (xi) removing the supernatant now containing the isolated progenitor cells.
An alternative method of separation which is based on sp*cific growth of pluripotent cells involves the following steps:
(i) a first step the whole human mammary secretion is subjected to centrifugation leaving a fat layer on top, a protein and carbohydrate rich supernatant beneath it, and at the bottom a pellet of cells.
(ii) in an optional second step, the cell pellet is washed in media. Preferably the cells are washed in RPMI media only.
/in) in a third step the cells of the cell pellet are plated onto a cell culture treated device in growth media and arc allowed to incubate. Preferably this incubation is carried out for 10-30 days, most preferably for 14-20 days.

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(iv) the cells axe harvested, i.e. they are detached from the support, preferably by trypsination. Subsequently the detached cells are washed, preferably using growth media solution.
(v) the harvested and washed cells are plated onto a reconstituted basement membrane preparation for growth, preferably up to confluence. In this last step, preferably a solubulizcd basement membrane preparation extracted from EHS mouse sarcoma is used, like e.g. Matrigel™ as available from BD Bioscienccs.
This method leads to cell cultures with a morphology which is typical for progenitor cells. The cells do not look like lactocytes and due to the growth media which is permissive for progenitor/stem cell/lactocyte growth, they cannot be bacterial cells.
The present invention furthermore relates to progenitor cells, which are preferentially pluripotent or multipotent progenitor (stem) cells, derived using a method as described above.
Additionally, the present invention relates to uses of such progenitor cells, for example for ex vivo, in vitro and/or in vivo applications. Without limiting the scope of the invention, such use may extend to the following specific examples or combinations thereof : creation of tissues or cells for the benefit of the mother and/or of the infant and/or of other individuals; subsequent gene therapy treatments or intrauterinc foetal treatments; generation of cells, tissue, glands or organs for the treatment of disease; subsequent cloning or scientific research; one or several of the group of the following purposes: clinical, diagnostic, bioengineering, lactoengineering, breast tissue regeneration, breast reconstructive surgery, breast cosmetic or enhancement surgery, exocrine gland tissue regeneration and/or surgery.
Further embodiments of the present invention are outlined in the dependent claims.
SHORT DESCRIPTION OF THE FIGURES
In the accompanying drawings preferred embodiments of the invention are shown in

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which:
Figure I shows the total mixed cell population of human milk following removal
of the fat and skim milk;
Figure 2 shows the stem cells (sc) following their isolation from a total milk cell
population. These cells have been cytospun onto microscope slides and stained with hacmatoxylin and eosin. Figure 2a) and 2b) show a single stem cell (sc). Figure 2c) shows a single stem cell still bound to the Dynabcads* (Db). The Dynabeads* (Db) are 4.5nM in size, thereby permitting the approximate sizing of the stem cells to be 6-7^M;
Figure 3 following purification of the stem cells from a total milk cell population,
the stem cells that are isolated can be cultured using a variety of culture conditions; this figure shows a single stem cell 1 day after being placed into culture;
Figure 4 after 1 to 2 weeks in culture, the stem cells isolated from a total cell
population begin to undergo cell division (mitosis); the arrow in this photo indicates a cell undergoing mitosis;
Figure 5 after several months in culture, these cells do not appear to have
differentiated into other cell types; this figure shows a stem cell after 2 months of culture which has been cytospun onto a microscope slide and stained with haematoxylin and eosin;
Figure 6 after 2 months in culture the stem cells have not differentiated; this figure
shows the stem cells isolated with the SSEA-4 (a) and Tra 1-60 (b) antibodies, following 2 months in culture; the cells were washed out of the culture medium and cytospun onto a microscope slide before being visualised on a confocal microscope with the SSEA-4 (a) and Tra 1-60 (b) antibodies conjugated to a fluorescently labelled secondary antibody (goat anti-mouse IgG conjugated to AlexaFluor-488).
Figure 7 shows the growth rate of isolated stem cells in culture; cells were seen to

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divide and proliferate over a period of 4 days; cells isolated with Dynabeads* and antibody SSEA-4 have shown an increase in total number of cells from approximately 50 cells per plate to 150 cells pcT plate in 4 days of culture; the central area of the plates was determined and a cruciform counting pattern was used; N=10 fields in each culture and numbers on individual days were simply totalled; the data shown in this Figure is the same experiment performed in triplicate.
Figure 8 shows a monolaycr of pluripotcnt cells as obtained from mammalian
secretion using an alternative procedure according to the invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
To determine whether a cell believed to be a stem cell is actually a stem cell, it has historically been necessary to undertake transplantation of the cells of interest into sub-lethally irradiated mice. If these transplanted cells subsequently locate and repopulate any organs of these mice, the cells in question have been considered to be pluripotent stem cells. However, more recently the identification of extracellular markers for pluripotcnt stem cells (for example Tra-1-60 and SSEA-4; Chcmicon International, Temccula, California, US) have enabled the identification of pluripotent (stem) cells without the prolonged process of transplantation.
After washing all cells out of human milk by a repeating the process of gentle centrifugation, aspiration of the supernatant and the rcsupension of the cells in a buffer or media several times, the stem cells were isolated from a total milk cell population (Figure 1) using dynabeads according to manufacturers specifications. As buffer Tris-buffcTed saline solutions (TBS) or phosphate buffered saline solutions (PBS) can be used. In particular, TBS-soIutions which are 10 mM in Tris, 150 mM in NaOH and which arc adjusted to have a pH around 7.4 can be used. In case of PBS-solutions, those maybe 1.1 mM KH2PO4, 140mM NaCl, 4.5mM NajHPO4.7H2O and 2.7mM NaCI, and may be adjusted to have a pH around 7.4. As media, Williams or RPMI (Roswell Park Memorial Institute) as available from Sigma Aldrich, US, under the product numbers

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R6504, R7755, R4130 or W4125, W4128, W1878, can be used. Also possible as media is RPMI from Gibco (CA, US) Cat. No 11875-093.
Stem cell-specific antibodies, e.g. Tra-1-60, Tra 1-81 and/or SSEA-4, both monoclonal, both e.g. available from Chemicon international, CHEMICON International, 28820 Single Oak Drive, Temecula CA 92590, were attached to the Dynabcads* for isolation of the progenitor cells. Also possible is the use of haematopoietic cells antibody CD 133 (cat* MAB1133) from R&D Systems, Inc CA, US.
Primitive haematopoietic stem and progenitor cells from peripheral blood have already been isolated using the immuno-magnctic cell separation principles. Research groups using monoclonal-antibody conjugated magnetic particles (CliniMACS System and Reagent AC133 from Miltcnyi Biotcc) have successfully isolated and cultured CD133 positive cells.
Any stem cell-specific antibody (inclusive of; SSEA-3, SSEA-1 and TRA 1-81 Oct-4, CD 133) are incubated with Dynabeads". Dynabeads* are available from Dynal AS, NO, and are small iron beads, that have an anti-mouse igG antibody attached to it via a small strand of DNA. Possible are for example Dynabcads* available under the name Dynal CD34. Incubation of the Dynabcads* is carried out for lh at room temperature prior to the Dynabeads being incubated with the cells isolated from milk for 15 minutes at 4°C.
Once the stem cells have bound to the Dynabcads which typically takes about 30 minutes to 1 hour, a magnet is attached to the side of the tube containing the cells/Dynabeads*. Dynabeads* are uniform, polystyrene-based, paramagnetic, beads with 4.5 urn in diameter. The Dynabeads' (with the stem cells attached) are attracted to the magnet, whereas the unbound cells arc not and remain in the supernatant. The supernatant is then removed leaving only the cells bound to the Dynabcads via the stem cell antibody. The cells bound to the Dynabcads* via the stem cell antibody arc removed by the addition of Dnase breaking the small strand of DNA. This is called releasing buffer and is part of the DynaJ kit 62500 U/ml (15000-20000U per vial quoted in the instructions). The Dynabeads* are removed by attaching the magnet once more to which the Dynabeads*, no longer attached to the stem cells, arc attracted. The

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supernatant now contains the stem cells which arc removed. These stem cells can now be used for any subsequent application as listed above/below.
The isolated progenitor (stem) cells can be cultured on mouse embryo fibroblast feeder cells in Knockout-Dulbccco's modified Eagle's medium. Typically cultivation can be carried out at a temperature of 37°C.
Examples of the use of feeder cells:
• Prolonged propagation of human embryonic stem cells is cunently achieved by
coculture with primary mouse embryonic fibroblasts serving as feeder cells.
• Acceleration of the formation of cultured epithelium using the sonic hedgehog
expressing feeder cells.
• Human adult marrow cells support prolonged expansion of human embryonic
stem cells in culture.
• Proliferation and maturation of embryonic stem cells into endothelial cells in an
in vitro model of vasculogenesis required OP9 feeder cells.
• Selective expansion and continuous culture of macrophages from adult pig
blood. Macrophages were selectively expanded and continuously cultured from
adult pig blood directly into the medium overlaying a feeder layer of STO mouse
fibroblasts.
• Establishment and characterization of a novel human immature
megakaryoblastic leukemia cell line, M-MOK, dependent on fibroblasts for its
viability. A novel fibroblast-dependent human immature megakaryoblastic
leukemia cell line (M-MOK) was established from the bone marrow of a girl
with acute megakaryoblastic leukemia, and its growth was determined to be
completely dependent on the presence of human embryonic lung-derived
fibroblasts, HEL-O.
• In vitro culture of embryonic disc cells from porcine blastocysts using foetal G30
porcine fibroblasts which had been previously irradiated as a feeder layer.

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• Acute lyinphocytic leukemias (ALL) cells of infants and children were found to preferentially survive in coculture with a cloned cell line of cndothelial adipose cells (14F1.1) from mouse bone marrow and exhibited extensive growth in the presence of the mouse stromal cells. These ALL cells were strictly dependent upon the mouse stromal clone 14F1.I and failed to proliferate in the absence of the endothelial adipocytcs or with a variety of feeder cells.
Cell growth
Cells isolated with Dynabcads* and antibody SSEA-4 have shown an increase in total number of cells from approximately 50 cells per plate to 150 cells per plate in 4 days (sec Figure 7).
Methods:
Cell preparation
150 ml of whole milk is spun for 15 minutes at 2000rpm to collect cells. Cell pellet is resuspended in approximately 4ml TBS 1% BSA and ccnlrifuged again for 10 minutes.
Final pellet is resuspended in 200^1 of RPMI 1% FCS.
Bead preparation
Beads are washed 3x in RPMI 1% foetal calf serum.
Beads coated with primary antibody at a concentration of lul in 500 u! TBS 1% BSA and incubated for 1 hour at room temperature with gentle mixing.
Coated beads separated and washed 3x in lml TBS 1% BSA and transferred to clean tube. Final wash in lml RPMI 1% FCS.
Cell isolation
200 \i\ cell preparation added to beads and incubated for 30min at approximately 100°C.

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Following incubation the unbound fraction is discarded.
200 ul RPMI 1% FCS is added to the cell and bead complex which is then gently pipetted to release further unbound matter.
To 200 ul of bead and cell complex add 5 ul releasing buffer and incubate for 15 minutes at 37°C with gentle mixing.
Vigorously pipette bead plus cell complex to help release cells.
Collect the 200 ul of isolated cell suspension and add to clean Eppcndorf.
This cell suspension can then be immediately introduced into the culture system.
Culture Method
Cells were seeded on collagen-coated culture plates.
Following a settling period the supernatant was carefully drawn off to remove contaminants and fresh media added. Cultures were then incubated in plates at 37°C in a 5% COj incubator with medium changes every two days.
Culture media
William's E medium supplemented with 10% fetal calf serum; 10'7M dexamethasone (Sigma); Gluiamine 2mmol/L ITS+ prcmix containing insulin (6.25ug/ml), transferrin (6.25ug/ml), sclcnious acid (6.25ng/ml), bovine serum albumin (1.25mg/ml) and linoleic acid (5.35yg/ml) from Becton-Dickinson, Bedford, MA. Penicillin / streptomycin 5,000 ng/ml Fungizone (25Oug/ml)
Antibodies
An example of some of the potential antibodies that can be used in this system arc; (All supplied by CHEMICON International, 28820 Single Oak Drive, Temccula CA 92590)
SSEA-I (Cat number MAB4301)

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SSEA-3 (MAB4303)
SSEA-4 (MAB4304)
TRA 1-60 (MAB4360)
TRA 1-81 (MAB4381)
haematopoietic cells antibody CD 133 (cattf MAB1133) from R&D Systems, Inc.
Buffers
TBS 1% BSA, lOmM Tris, 150 mM NaOH
RPMI 1%FCS
PBS 1.1 mM KH2PO Following purification of the stem cells from human milk, using the extracellular markers for stem cells (Tra-1-60 and SSEA-4) bound to Dynabeads* we cytospun the stem cells onto microscope slides and stained the stem cells using haematoxylin and eosin (Figure 2). Using these same purified stem cells we have been able to culture the cells (Figure 3) and demonstrate that these cells do undergo cell division (Figure 4). Following prolonged culture, we have been able to stain these cells using haematoxylin and cosin (Figure 5), and have demonstrated that these cells remain stem cells as they continue to bind the stem cell antibodies (Tra-1-60 and SSEA-4) following several months of culture, as visualised by confocal microscopy (Figure 6). Growth of the isolated cells was also verified, see Figure 7.
An alternative way of isolation/growth of the pluripotent cells is using a specific combination of growth media by means of which it is possible to grow a monolayer of pluripotent cells that clearly do not look like lactocytes (see Fig. 8). Indeed the grown cells in culture are physically identical to for example liver stem cell cultures. Further

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classification of these cells by the cellular markers they express and by their gene activity is possible.
This alternative way of isolation or selective growth can be carried out as follows: Preparation of Matrigel™ coated plates:
The Matrigel (commercially available reconstituted basement membrane, BD Matrigel™, BD Bioscicnccs rcf-354234) was allowed to defrost slowly on ice. Pipette tips and culture plates were cooled and kept on ice prior to use. 50ul of the thawed Matrigel was aliqouted and spread with a pipette tip into each of the wells of a 24 well flat bottom microplatc and allowed to incubate on ice for 30 minutes. Following this the plates were further incubated for 30 minutes at 37 degrees centigrade in order to set the gel. Once set lml of growth media was aliquoted to each well and the plate returned to the incubator until required (no longer than 5 days).
Growth and preparation of cells
Freshly expressed hind milk (approximately 50ml, hind milk is human breastmilk that is high in fat content and creamy in color, it provides the bulk of the baby's calories and is most responsible for weight gain) was centriftjged to pellet the cells (details as given above).
The cell pellet was washed 2x in RPM1 media (RPMI Medium 1640, Gibco ref-108-36) only.
Cells were then plated onto cell culture treated plastic culture dishes in growth media (Cell culture growth media composition: RPMI 1640; Fetal Bovine scrum 20*/»; Insulin 5ug/ml; Cholera toxin 50ng/ml; Hydrocortisonc 0.5ug/ml; Penicilin streptomycin fungjzone 2x) and allowed to incubate for approximately 14-20 days.
After this period plates showing large colonies and approaching confluence were selected and the cells harvested by trypsinization (Trypsin-EDTA lx, JRH Bioscicnce

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ref=59218).
The harvested cells were washed Ix in growth media and plated onto Matngel™ coated plates at a density approximately 1500 cells/ml.
After around 14 days patches of confluent cells can be observed.
Description
The morphology is typically epithelial in that confluent sheets of cells develop and the cells within the colonies are closely associated. The fact that breastmilk derived cells cultured in this manner will proliferate in primary culture and form extensive circular colonies with well defined borders is strongly indicative of cells with progenitor like qualities. That the cells appear undiffercntiated and many can be observed to have a large nucleus to cytoplasm ratio is even more evidence for a progenitor like identity.
Specific materials used
Cholera toxin (LIST BIOLOGICAL LABORATORIES ref-lOlB)
Fetal Bovine serum (Invitrogen rcf-10099141)
Hydrocortisone (Sigma ref-HO135)
Insulin (Sigma ref-19278)
Pcncillin/Strepomycin Fungizone (Scott Scientific ref-17.745E)
MICROPLATE (IWAKJ rcf- 3820-024)
Cell Culture Dish (Coming ref-430165)
Due to the plasticity of the pluripotent stem cells, these isolated cells can be utilised for a multitude of different applications. For example these cells can be;
• used to create tissues for the benefit of the mother and infant (and potentially

WO 20OS/W1696 PCT/CH 2004/000738
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other individuals), including gene therapy treatments, intrauterine foetal treatments, and the generation of cells, tissue, glands, or organs for the treatment of disease. This includes their use in scientific research, clinical, diagnostic or commercial applications. This can also include the generation of biological compounds including cells, cellular compartments, cellular secretions, cell isolates, nucleotides, deoxyribonucleic acids, amino acids, proteins, glycoproteins, carbohydrates, lipids, hormones, growth factors, and cytokines. Furthermore, this can include the generation of cells as a precursor, or as a consequence, of the generation of tissue, glands or organs for the treatment of disease, tissue regeneration, body enhancement, or cosmetic applications for the following tissues; Olfactory, Auditory, Optical, Lymphatic, Immune, Haematopoietic, Endocrine, Exocrine, Bowel, Gastrointestinal, Pcycrs Patches, Islets of Langerhans, Skeletal, Muscle, Connective, Vascular, Blood, Skin, Hair, Nails, Mammary, Brain and Central Nervous System, Liver, Heart, Lung, Kidney, Bone, Pancreas, Reproductive.
• stored for future. The subsequent use of these stem cells, or cells differentiated
or differentiated could include storage of these stem cells for future use as
outlined below. This includes their storage for use in scientific research,
clinical, diagnostic or commercial applications.
• used for cell culture, whether this be for propagation of these same stem cells, or
for the differentiation or dedifferentiation of these stem cells into another cell
type. This includes their use in scientific research, clinical, diagnostic or
commercial applications.
• used for cloning. The subsequent use of these stem cells, or cells differentiated
or dedifferentiatcd from these stem cells could be used to generate clones,
whether embryonic or whole animal. This includes their use in scientific
research, clinical, diagnostic or commercial applications.
• used for scientific research. The subsequent use of these stem cells, or cells
differentiated or dediffcrentiated from these stem cells could be used in scientific

WO 200.V061696 PCT/CH20O4/UO0738
17
research. This can include the generation of biological compounds including cells, cellular compartments, cellular secretions, cell isolates, nuclcotides, dcoxyribonucleic acids, ami no acids, proteins, glycoproteins, carbohydrates, lipids, hormones, growth factors, and cytokincs. In addition this could include the generation of cells as a precursor, or as a consequence, of the generation of tissue, glands or organs for the treatment of disease, tissue regeneration, body enhancement, or cosmetic applications for the following tissues; Olfactory, Auditory, Optical, Lymphatic, Immune, Haematopoietic, Endocrine, Exocrine, Bowel, Gastrointestinal, Peycrs Patches, Islets of Langerhans, Skeletal, Muscle, Connective, Vascular, Blood, Skin, Hair, Nails, Mammary, Brain and Central Nervous System, Liver, Heart, Lung, Kidney, Bone, Pancreas, Reproductive.
• used for clinical, diagnostic or commercial applications. The subsequent use of
these stem cells, or cells differentiated or dedifferentiated from these stem c«lls
could be used in clinical, diagnostic or commercial applications. This can
include the generation of biological compounds including cells, cellular
compartments, cellular secretions, cell isolates, nuclcotides, deoxyribonucleic
acids, amino acids, proteins, glycoproteins, carbohydrates, lipids, hormones,
growth factors, and cytokines. In addition this could include the generation of
cells as a precursor, or as a consequence, of the generation of tissue, glands or
organs for the treatment of disease, tissue regeneration, body enhancement, or
cosmetic applications for the following tissues; Olfactory, Auditory, Optical,
Lymphatic, Immune, Haematopoietic, Endocrine, Exocrine, Bowel,
Gastrointeitinal, Pcyers Patches, Islets of Langerhans, Skeletal, Muscle,
Connective, Vascular, Blood, Skin, Hair, Nails, Mammary, Brain and Central
Nervous System, Liver, Heart, Lung, Kidney, Bone, Pancreas, Reproductive.
• used for bioengineering. The subsequent use of these stem cells, or cells
differentiated or dediffereniiatcd from these stem cells could be used to generate
any other cell type in the human body. These cells, tissues, or organs could then
be used for cosmetic/reconstructive surgery, organ/tissue transplantation or the

WO 200W061696 PCT/CH20O4/000738
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generation of cells/tissue/organs for a third party. This can include the generation of biological compounds including cells, cellular compartments, cellular secretions, cell isolates, nucleotides, deoxyribonuclcic acids, amino acids, proteins, glycoproteins, carbohydrates, lipids, hormones, growth factors, and cytokines. In addition this could include the generation of cells as a precursor, or as a consequence, of the generation of tissue, glands or organs for the treatment of disease, tissue regeneration, body enhancement, or cosmetic applications for the following tissues; Olfactory, Auditory, Optical, Lymphatic, Immune, Haematopoietic, Endocrine, Exocrinc, Bowel, Gastrointestinal, Peycrs Patches, Islets of Langcrhans, Skeletal, Muscle, Connective, Vascular, Blood, Skin, Hair, Nails, Mammary, Brain and Central Nervous System, Liver, Heart, Lung, Kidney, Bone, Pancreas, Reproductive.
• used for lactoengineering. The subsequent use of these stem cells, or cells
differentiated or dedifferentiated from these stem cells could be used to generate
biological compounds of milk including cells, cellular compartments, cellular
secretions, cell isolates, nucleotides, deoxyribonucleic acids, amino acids,
proteins, glycoproteins, carbohydrates, lipids, hormones, growth factors, and
cytokines.
• breast tissue regeneration. The subsequent use of these stem cells, or cells
differentiated or dedifferentiated from these stem cells could be used to generate
breast tissue.
• breast reconstructive surgery. This regenerated tissue as above could then be
used for reconstructive breast surgery.
• breast cosmetic surgery This regenerated tissue as above could then be used for
cosmetic breast surgery.
• exocrinc gland tissue regeneration and/or surgery. The subsequent use of these
stem cells, or cells differentiated or dedifferentiated from these stem cells could
be used to generate exocrine gland tissue, which in rum, could be used for the
regeneration or replacement of exocrine glands.

WO 2005/06)696 PCT/CH2004/000738
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the generation of biological compounds including cells, cellular compartments, cellular secretions, cell isolates, nuclcotides, dcoxyribonucleic acids, amino acids, proteins, glycoproteins, carbohydrates, lipids, hormones, growth factors, and cytokines.

WO 2005/061696 PCT/CH20O4/O0O7J8
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CLAIMS
1. A method for isolating progenitor cells from a human body, inclusive of all cells
with stem cell-like characteristics, wherein such cells are directly or indirectly
derived from human mammary secretion, be it colostrum, mature milk, or dry
period secretion from males or females, of said human body during at least one
of the following periods: non-pregnant period, pregnant period, lactating period,
involuting period.
2. A method according to claim 1, wherein the progenitor cells are pluripotcnt or
multipotent.
3. A method according to claim I, wherein said progenitor cells arc isolated from
the mammary secretion in that noncellulax parts of the mammary secretion arc
separated from the cellular parts.
4. A method according to claim 3, wherein non-pluripotent or non-multipotcnt cells arc removed from cellular parts.
5. A method according to any of the preceding claims, wherein secretory epithelial
cells, leucocytes and in particular nonhuman cells like bacterial cells are
removed from the mammary secretion.
6. A method according to any of the preceding claims, wherein the mammary
secretion during lactating periods is used for the isolation of the progenitor cells,
and wherein the mammary secretion during particular stages of mammary
secretion such as: after beginning of individual feeding; versus end of individual
feeding; lactation phase; preferably early lactation.

WO 2005/061696 PCT/CH20O4/0OO738
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7. A method according to any of the preceding claims, wherein magnet beads are used to ihc isolation of the progenitor cells.
8. A method according to any of the preceding claims, wherein in a first step cellular components axe washed out of the mammary secretion, in a second step said cellular components are stained with antibodies to the progenitor cell markers, and in a third step the progenitor cells arc separated from the other cells directly or indirectly by means of the attached antibodies.
9. A method according to claim 8, wherein the antibody-stained progenitor cells are
attached to beads, preferably small iron beads, and wherein the progenitor cells
are extracted by means of the beads, preferably in case of small iron beads by
using a magnet, and wherein subsequently the beads as well as if need be the
antibodies are removed from the progenitor cells.
10. A method according to claim 9, wherein removal of the beads is effected by
means of enzymes selected from the following group: Dnase, Proteinase, Rnase.
11. A method according to any of the preceding claims, wherein the progenitor cells
are cultured without using a fibroblast feeder layer, in particular without using a
mouse fibroblast feeder layer.
12. A method according to any of the preceding claims, wherein in
(i) a first step the whole human mammary secretion is subjected to centrifugation leaving a fat layer on top, a protein and carbohydrate rich supernatant beneath it, and at the bottom a pellet of cells;

WO 2005/061696 PCT/CH20O4/OOO738
22
(ii) in a second step fat fraction and supernatant are removed;
(iii) in a third step a buffer, such as, but not limited to, phosphate buffered saline,
tris buffer saline, TBS and/or PBS, or media, such as, but not limited to,
Williams media or RPMI Media, is added and the cells are rcsuspended in the
buffer / media and centrifuged as before, preferentially repeating this process 3
or 4 times, leaving a substantially pure cell pellet;
(iv) and in a fourth step the progenitor cells are separated from the cell pellet.
13. A method according to any preceding claim, wherein a oell pellet is generated from the human mammary secretion, and subsequently the following separation steps are used:
(v) the cell pellet is suspended in media, preferentially in RPMI media containing foetal calf (bovine) serum,
(vi) this suspension is incubated with magnetic beads which have before been incubated with progenitor, preferentially stem cell-specific antibodies, like anti-mouse IgG antibodies, which antibodies arc attached to the magnetic beads via a small strand of DNA, wherein the incubation of the cell suspension is preferentially carried our for 15 minutes at 4°C;
(vii) once the progenitor cells have bound to the magnetic beads a magnet is attached to the tube containing the cells/beads, thus attracting the progenitor cells connected with the beads to the magnet, whereas unbound cells are not and remain in the supernatant;
(viii) removing the supernatant leaving only the progenitor cells bound to the beads via the progenitor cell antibody.
14. A method according to claim 13, wherein subsequently, the following steps are used:
(ix) progenitor cells bound to the beads via the stem cell antibody arc removed by an appropriate cleavage means, preferentially, in case of the antibody being

WO 2005/061696 PCT/CH20O4/0O0738
23
attached to the beads via small strand of DNA, a by means of addition of a
Dnase,
(x) the beads arc removed by attaching the magnet once more such that the
beads, no longer attached to the stem cells, are attracted to it;
(xi) removing the supernatant now containing the isolated progenitor cells.
15. A method according to any of claims 1-12, wherein the cells are separated from human mammary secretion by centrifugation, subsequently incubated in a growth media that is permissive for progenitor/stem cell/lactocyte growth.
16. A method according to claim 15, wherein in
(i) a first step the whole human mammary secretion is subjected to centrifugation leaving a fat layer on top, a protein and carbohydrate rich supernatant beneath it, and at the bottom a pellet of cells;
(ii) in a second step, the cell pellet is washed in media, preferably in RPMI media only
(iii) in a third step the cells of the cell pellet are plated onto a cell culture treated device in bacteriocidal and/or fungicidal growth media and are allowed to incubate, preferably for 10-30 days, most preferably for 14-20 days,
(iv) the cells are harvested, preferably by trypsination, and washed, preferably using growth media
(v) the harvested cells are plated onto a reconstituted basement membrane preparation for growth preferably up to confluence.
17. A method according to claim 16, wherein in step (v) a solubulized basement
membrane preparation extracted from EHS mouse sarcoma is used, as e.g.
Matrigel™.

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18. Progenitor cells, preferentially pluripotent or multipoint progenitor cells,
derived using a method according to any of the preceding claims 1 through 17.
19. Use of pluripotent or multipotcnt progenitor cells as derived using a method
according to any of the claims 1-17 for ex vivo, in vitro and/or in vivo
applications.
20. Use according to claim 19, to create tissues or cells for the benefit of the mother and/or of the infant and/or of other individuals.
21. Use according to claim 17 or 20, including subsequent gene therapy treatments or intrauterine foetal treatments.
15 22. Use according to claim 19-21, for the generation of cells, tissue, glands or organs for the treatment of disease.
23. Use according to any of the claims 19-23, for subsequent cloning or scientific research.
24. Use according to any of the claims 19-23, for one or several of the group of the following purposes: clinical, diagnostic, bioengineering, lactoengincering, breast tissue regeneration, breast reconstructive surgery, breast cosmetic or enhancement surgery, exocrine gland tissue regeneration and/or surgery.

Documents:

01326-kolnp-2006-abstract.pdf

01326-kolnp-2006-assignment.pdf

01326-kolnp-2006-claims.pdf

01326-kolnp-2006-correspondence other.pdf

01326-kolnp-2006-correspondence others-1.1.pdf

01326-kolnp-2006-correspondence-1.1.pdf

01326-kolnp-2006-description complete.pdf

01326-kolnp-2006-drawings.pdf

01326-kolnp-2006-form 1.pdf

01326-kolnp-2006-form 3.pdf

01326-kolnp-2006-form 5.pdf

01326-kolnp-2006-form-18.pdf

01326-kolnp-2006-form3-1.1.pdf

01326-kolnp-2006-international publication.pdf

01326-kolnp-2006-international search report.pdf

01326-kolnp-2006-pct form.pdf

1326-KOLNP-2006-(17-10-2012)-CORRESPONDENCE.pdf

1326-KOLNP-2006-(19-11-2012)-ANNEXURE TO FORM 3.pdf

1326-KOLNP-2006-(19-11-2012)-CORRESPONDENCE.pdf

1326-KOLNP-2006-(25-09-2008)-FORM 13.pdf

1326-KOLNP-2006-ABSTRACT.pdf

1326-KOLNP-2006-CANCELLED DOCOMENT.pdf

1326-KOLNP-2006-CLAIMS.pdf

1326-KOLNP-2006-DESCRIPTION COMPLATE.pdf

1326-KOLNP-2006-REPLY TO EXAMINATION REPORT.pdf

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Patent Number

256735

Indian Patent Application Number

1326/KOLNP/2006

PG Journal Number

30/2013

Publication Date

26-Jul-2013

Grant Date

23-Jul-2013

Date of Filing

18-May-2006

Name of Patentee

CARAG AG

Applicant Address

BAHNHOFSTRASSE 9, CH-6340 BAAR,

Inventors:

#	Inventor's Name	Inventor's Address
1	GREGAN, MARK, DEREK	19 CARRICK ST., WOODLANDS, WA 6018
2	HARTMANN, PETER, EDWIN	21 JOHN STREET, GOOSEBERRY HILL, WA 6076

PCT International Classification Number

C12N 5/06

PCT International Application Number

PCT/CH2004/000738

PCT International Filing date

2004-12-15

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	PCT/CH2003/000846	2003-12-23	Switzerland