WO2008080200A1 - Procédé d'obtention de cellules souches - Google Patents

Procédé d'obtention de cellules souches Download PDF

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WO2008080200A1
WO2008080200A1 PCT/BR2006/000309 BR2006000309W WO2008080200A1 WO 2008080200 A1 WO2008080200 A1 WO 2008080200A1 BR 2006000309 W BR2006000309 W BR 2006000309W WO 2008080200 A1 WO2008080200 A1 WO 2008080200A1
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fact
cells
cell
stem cells
diseases
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PCT/BR2006/000309
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Irina Kerkis
Alexandre Kerkis
Mirian Akemi Furuie Hayashi
Humberto F. Cerruti
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Irina Kerkis
Alexandre Kerkis
Mirian Akemi Furuie Hayashi
Cerruti Humberto F
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Priority to PCT/BR2006/000309 priority Critical patent/WO2008080200A1/fr
Publication of WO2008080200A1 publication Critical patent/WO2008080200A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0664—Dental pulp stem cells, Dental follicle stem cells

Definitions

  • the present invention refers to a process of obtainment of adult stem cells presenting embryonic stem cells characteristics, from postnatal or adult tissues, to adult stem cells obtained by said process that present embryonic stem cells characteristics, to an advantageous process of stem cells differentiation, and to differentiated cells obtained from said process.
  • the present invention also refers to the numerous possibilities of therapeutic and non-therapeutic, biotechnological and pharmaceutical application of said cells, either differentiated or undifferentiated, resulting from the development achieved by the inventors.
  • the embryonic stem cell (ES) strains are undifferentiated cells, originated from blastocysts inner mass, which main characteristic is the pluripotency (Evans, M., and Kaufman, M. (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154-6).
  • said ES cells when reintroduced into a blastocyst, said ES cells are capable to resume their normal development and have the ability to colonize distinct embryo tissues, including the germinative strain.
  • Highly pluripotent ES cells are only those with the ability to colonize embryo germinative strains. When cultivated under specific conditions, ES cells can be maintained in its undifferentiated state through multiple cell divisions.
  • Ebs embryoid bodies
  • Morphological, immunohistochemical and molecular analyses allow finding a large variety of embryonic strains in the Ebs - hematopoietic, neuronal, endothelial, cardiac and muscular (Ling, V., and Mau, S. (1997) In vitro differentiation of embryonic stem cells: immunophenotypic analysis of cultured embryoid bodies. J. Cell Physiol. 171 , 104-5).
  • ES cells Those properties of ES cells allow their use as an in vitro model of precocious embryonic development.
  • the use of these cells permit the study of cell differentiation mechanisms, X chromosome inactivation process and effects of toxic and biologically active substances on the process of in vitro embryonic development.
  • ES cells Moreover, the capacity of ES cells to differentiate themselves into any type of tissue represents an enormous potential for medical applications. When induced to differentiation in vitro into a determined cellular type, under specific conditions, they represent an unlimited source of tissues for transplants and treatment of diseases. An important step in this direction was the establishment of ES cells strains: both human, (Thompson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknits, M.A., Swiegiel, J.J., Marshall, V.S., Jones, J.M. (1998) Embryonic stem cell lines derived from human blastocysts.
  • stem cell populations obtained through the usual methods do not allow the obtainment of homogeneous cultures, i.e., constituted solely by stem cells.
  • stem cells obtained through the known methods generally present a limited capacity of differentiation, wherein the strains known on the prior art differentiate only into a restricted number of tissues or cellular types, specific for each cell strains. As such, stem cells obtained through the known processes show limited plasticity.
  • hematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial membrane. Nature, 428:664-668).
  • US Patents 6,569,427 and 6,605,275 describe the isolation and the preservation of fetal and neonatal hematopoietic stem cells and blood progenitor cells and the use of these cells upon thawing for therapeutic purposes. Particularly, they refer to the therapeutic use of said fetal or neonatal stem cells in the hematopoietic (or immune) reconstitution.
  • the hematopoietic reconstitution with these cells is valuable for the treatment or prevention of several diseases and disorders such as anemias, malignant diseases, autoimmune disorders and several immune dysfunctions and deficiencies.
  • progenitor cells and fetal or neonatal stem cells that contain heterologous sequences may be used for hematopoietic reconstitution in gene therapy.
  • these cells that may be cryopreserved and thawed can be used for autologous reconstitution (for donor own or self use).
  • the cells present a limitation in the capacity of differentiation. Additionally, the process requires performing a series of selection stages of the cells that will be used.
  • US Patent 6,596,274 describes biologic materials containing bone marrow stem cells partially or completely differentiated into conjunctive tissue cells and a hyaluronic acid ester matrix. It is also described a two-component biologic material where the first can alternatively be (1) an autologous or homologous culture of bone marrow stem cells partially or completely differentiated into a specific conjunctive tissue cellular strains or (2) a pure extracellular matrix free of any cellular components secreted by a specific conjunctive tissue cellular strains, and a second component containing a biocompatible and biodegradable tridimensional matrix, constituted by a hyaluronic acid ester with an estherification grade between 25 and 100%.
  • the specific tecidual strains are selected from the fibroblasts, osteoblasts, myoblasts, adipocytes, chondrocytes and endothelial cells.
  • the biologic material is adequate for use as a dermal replacement for cutaneous lesions, as well as, for the repair of damages to the conjunctive tissue. As in the previous document cited, it results in a cellular population with a limited differentiation capacity.
  • US Patent 6,638,763 describes methods for obtainment and isolation of neural stem cells in mammals. Through the use of a new culture strategy, unknown neural progenitor cell populations have been found in mammal's adult brains. Through a limited cell-to-cell contact, the dissociated adult brain generates at least two types of cellular aggregates.
  • These aggregates or stem/precursor cell clones may be generated from adult brain tissues after a significant post mortem time interval. Both neurons and glias can be generated from a culture of these cells and these cells can also survive the transplant into mammal's adult brains. However, the obtainment of said cells is limited to the recovery of material from dead donors and, therefore, restricted to the heterologous transplant, which viability still depends on the immunocompatibility between the donor and the patient receptor.
  • the main sources of stem cells currently used in medicine are the bone marrow and the umbilical cord.
  • the isolation of a pure population of pluripotent stem cells and in large amounts is difficult, being required alternative sources for the isolation of highly pluripotent and homogeneous stem cells, which will significantly increase the treatment efficacy of several diseases.
  • Dental pulp stem cells have been isolated from humans as well as from rats, showing the ability to differentiate in vivo into bone, cartilage and dentin. So, the stem cells obtained as described present mesenchymal characteristics, determined by the isolation and amplification processes used, resulting in a limited capacity of differentiation (Yamamura, T.J (1985) Differentiation of pulpal cells and inductive influences of various matrices with reference to pulpal wound healing. Dent. Res. 64 Spec No: 530-40; Caplan, A.I. (1991) Mesenchymal stem cells. J. Orthop. Res. 9:641-650; Mann, L.M., Lennon, D.P. and Caplan, A.I.
  • PNAS v.97, 25:13625-13630; Gronthos, S., Brahim, J., Li, W., Fisher, L.W., Cherman, N., Boyde, A., Den Besten, P., Gehron Robey, P., and Shi, S. (2002) Stem cell properties of human dental pulp stem cells. J. Dent. Res. 81:531-535; Miura, M., Gronthos, S., Zhao, M., Lu, B., Fisher, LW., Gehron Robey, P., and Shi, S. (2003) SHED: Stem cells from human exfoliated deciduous teeth. PNAS 100,:5807-5812).
  • US Patent 6,767,740 describes methods and instruments for the recovery of dental pulp and stem cells, wherein the obtained cells can be propagated, expanded and, subsequently, used in the repair or regeneration of body tissues in therapeutic treatment and other medical purposes.
  • the method disclosed comprises a maceration step and results in a heterogeneous population of cells with mesenchymal characteristics.
  • Another patent document that reports the obtainment of adult stem cells isolated from human dental pulp is identified as US 20040058442.
  • the inventors describe an adult stem cells culture, isolated from human dental pulp, where said cells can be differentiated into dentin/pulp tissues becoming, therefore, useful in the dentistry area.
  • the document describes a process that comprises the dissociation of the dental pulp cells. However, it neither reveals a pure stem cells population nor cites embryonic stem cells properties.
  • the development achieved by the inventors allows perform a process presenting said characteristics, a practical and simple way to isolate, from postnatal or adult tissue, homogeneous populations of adult stem cells that present characteristics of embryonic stem cells, capable of differentiating into several cellular types even without the presence of inducers, resulting in the possibility of autologous and/or heterologous treatment, using not only differentiated cells obtained in the absence of usually employed inducer factors, but undifferentiated stem cells showing embryonic stem cells characteristics, as well.
  • the present invention refers to a process for obtainment of stem cells that show, besides other advantages, the characteristics of embryonic stem cells, even though they are originated from postnatal or adult tissues, as a result of an innovative set of steps.
  • Another object of the present invention is to provide adult stem cells presenting the characteristics of embryonic stem cells, obtained from said process.
  • the present invention refers to the process for differentiation realized from said cells and concentrates containing these cells, as well as, to the differentiated cells obtained from said process and concentrates containing these cells.
  • an aspect to be stressed in the development reached by the inventors concerns the methods for treatment and/or prevention of diseases that comprises the application, in patients, of therapeutically effective amount of said cells or concentrates, differentiated or not, that result in numerous possibilities of prevention and treatment of diseases, as well as for the use in the development of new drugs.
  • the present invention also refers to the use of said cells and/or cell concentrates, differentiated or not, in the therapeutic and/or preventive treatment of diseases.
  • Another object of the present invention is the biotechnological use of the cells and concentrates, differentiated or not, such as in the cellular modification, tissue engineering, genetic or cellular therapy as well as in pharmaceutical research.
  • the present invention also concerns the cell banks comprising adult stem cells presenting characteristics of embryonic stem cells, obtained from the process of the present invention or concentrates containing these cells. Said cells have the capacity to maintain the characteristics herein described even after cryopreservation and thawing, allowing its use in a cell bank.
  • Figure 1 illustrates the process for growing adult stem cells derived from human dental pulp, wherein in A the growth kinetics of the DL-1 , DL-2, DL-3 e DL-4 cell strains are illustrated, as a function of the number of passages; panel B shows the FACS (fluorescence-activated cell sorter) analysis of the expression of PCNA antigen (proliferating cell nuclear antigen) in DL-3 strain, and panel C is the negative control.
  • FACS fluorescence-activated cell sorter
  • Figure 2 illustrates the expression profile of markers for pluripotent, endothelial and hematopoietic stem cells, observed in different strains of adult stem cells derived from human dental pulp.
  • panel A the culture of adult stem cells from human dental pulp, observed through PC is shown; the immunofluorescence analysis of these cells using the following antibodies: anti-SSEA-4 (B), for strain DL-2; anti-Oct-4 (C), for strain DL-1; anti- SSEA-3 (D) for strain DL-3; anti-TRA-1-60 (E) for strain DL-3; and anti-TRA-1- 81 (F) for strain DL-4, are also shown.
  • Positive reaction to Oct-4 (G) and SSEA- 1 (H) observed in mouse embryonic stem cells (mES cells) is also shown.
  • Figure 3 shows the myogenic differentiation of adult stem cells from human dental pulp.
  • the PC images of the formation of myoblast fusion (A) and multinuclear syncytium (B) by differentiation of DL-1 cells strain are also shown.
  • panel C the spontaneous contraction (*) of mature muscle fibers (PH) is shown.
  • panel D the Z-discs formation, observed by PC, is illustrated.
  • the EF images of skeletal muscle cells differentiated from the DL-1 strain, using antibodies anti-titin (E) and anti-alpha-actinin (sarcomeric) (F) are shown.
  • Figure 4 illustrates the chondrogenic differentiation of adult stem cells from human dental pulp.
  • a to C distinct stages in the cartilage formation, observed by PC in living cultures of adult stem cells of human dental pulp (hDPs) are shown; (*) indicates undifferentiated cells.
  • panel D the specific histochemical staining (toluidine blue) for differentiated chondroblasts, visualized by PC, is shown.
  • panel E the synthesis of proteoglycan chondroitin sulfates (aggrecans) into differentiated chondroblasts, evidenced by anti-ACSP antibodies (anti-smooth muscle actin), is shown.
  • the arrows indicate the secretion granules from aggrecans of differentiated chondroblasts visualized by EF.
  • Figure 5 shows the neuronal differentiation of the adult stem cells from human dental pulp (hDPs) and adherent SLS derived from adult stem cells from human dental pulp (hDPs).
  • panel A 1 a three-day living cell culture evidencing a positive immuno-labeling for nestin (B) and ⁇ -tubulin III (C) is illustrated.
  • Panel D shows the superposition of images B and C.
  • panel E a 7- days after plated SLS culture is illustrated. The upper layer of cells is composed by neurons indicated by the arrows (PC).
  • panel F the cells positive for GFAP (glial fibrilic acidic protein) and in panel G cells positive for ⁇ -tubulin III are shown.
  • panels H to K the different stages in the neuron formation, evidenced by the positive reaction to ⁇ -tubulin III (EF), are shown.
  • the magnification for E and G is 350X; for F is 600X and for H to K is 800X.
  • Figure 6 illustrates the presence of the human gene GBA observed by PCR analysis in different organs of the mouse, three months after the injection of DL-3 cell strain.
  • molecular marker 100 bp, Invitrogen
  • human DNA 3, strain DL-3 DNA ; 4, mouse DNA, as negative control; 5, lung (intraperitoneal); 6, spleen (intraperitoneal); 7, kidney (subcutaneous); 8, muscle (subcutaneous).
  • the object of the present invention is a process for obtainment of stem cells comprising the following steps: a) obtainment of stem cells from postnatal or adult tissues; b) transfer into a vessel containing appropriate medium, wherein said transfer is performed without cell dissociation; c) growing and expansion of cells.
  • tissue obtained from dental pulp, bone marrow, blood, nervous tissue, umbilical cord, muscle or skin.
  • postnatal tissue means tissue recovered from fetuses or newborns and by "adult tissue” is meant tissue obtained after the postnatal period.
  • the stem cells are particularly extracted from dental pulp.
  • the material is isolated from deciduous or adult teeth, but preferably isolated from deciduous teeth, from the first dentition, usually from a child up to 10 years-old, by removal of the tooth apical section.
  • step (a) After the extraction in step (a), the cells are washed, preferably with a sterile buffered saline solution, with or without antibiotics, according to protocols known to the skilled in the art, after which step (b) is performed.
  • appropriate medium in step (b) refers to culture media for stem cells capable of promoting the isolation of a pure and homogeneous population of stem cells that present characteristics of embryonic stem cells, as for example DMEM/F12 (Dulbecco's modified Eaglee's medium/Ham's F12, 1 :1, Invitrogen, Carlsbad, CA), also encompassing possible alternatives, variations and upgradings equivalent to this cell culture medium.
  • DMEM/F12 Dulbecco's modified Eaglee's medium/Ham's F12, 1 :1, Invitrogen, Carlsbad, CA
  • Said medium is preferably supplemented with serum and, in accordance with the particular embodiment of the process includes at least 5% of serum, and more typically about 15% of serum.
  • said serum is from bovine origin, more particularly bovine fetal serum, although synthetic and non-synthetic serums, from human and other animals may also be employed, as well as other synthetic or natural reagents, including mixtures thereof, that allow the isolation of said cells
  • the cell culture medium, in step (b), may additionally contain antibiotics and/or amino acids.
  • the antibiotics used well-known to those skilled in the art, may in particular be penicillin and streptomycin.
  • amino acids useful to perform the present invention may be cited glutamine and other non-essential amino acids and mixtures thereof.
  • the culture, in step (c) of the present invention is a continuous culture, wherein cells can be cultivated through several passages. According to an embodiment of the present invention, after a period of 1-3 weeks of culture, the cells are dissociated and transferred to vessels that contain the same culture medium of step (b). According to a preferred embodiment, the cell culture of the present invention is maintained semiconfluent, in order to prevent cell differentiation.
  • semiconductor it is understood a culture that is not so dense to the point where the cells become in substantial contact with one another, as it happens in high density confluent cultures.
  • the cell dissociation may be realized through mechanical or enzymatic means.
  • Said dissociation should preferably be realized by enzymatic means, more particularly by means of trypsinization, preferably using a solution of around 0.05% of trypsin in a solution containing ethylenediaminetetraacetic acid (EDTA).
  • EDTA ethylenediaminetetraacetic acid
  • the cells may be frozen for preservation of the culture.
  • the stem cells in the present invention admit its cryopreservation in the undifferentiated state.
  • the cells are re-suspended in an appropriate medium.
  • the medium contains from 10 to 20%, preferably 20% of serum, from 70 to 80%, preferably 70% of DMEM and from 5 to 10%, preferably 10% of dimethyl sulfoxide (DMSO).
  • the temperature is slowly and gradually decreased, at a rate of approximately 1 0 C per minute, down to a final temperature of around -7O 0 C to -8O 0 C, and the cells are transferred, after approximately 24 hours, to a tank containing liquid nitrogen.
  • the cell culture obtained in accordance with the process of the present invention is a pure culture of stem cells with characteristics of embryonic stem cells.
  • the differentiation potential of clonal cell strains varied significantly, so that only % of the clones were capable of differentiating into dentin-like tissues when transplanted into immunocompromised mice, and only about 5% of the cells were able to differentiate in vitro into adipocytes (Gronthos, S., Mankani, M., Brahim, J., Gehron Robey, P., and Shi, S. (2000) Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. PNAS, v.97, 25:13625-13630; Miura, M., Gronthos, S., Zhao, M., Lu, B., Fisher, LW., Gehron Robey, P., and Shi, S. (2003) SHED: Stem cells from human exfoliated deciduous teeth. PNAS 100:5807-5812).
  • the cells obtained with the process herein described, present in their undifferentiated state, a group of cellular surface markers typical of embryonic cells.
  • the patent document US 2004/0180347 describes a markers system for characterization of human pluripotent embryonic stem cells, their advantages and applications.
  • Said cells express in their undifferentiated state a group of cell surface embryonic markers (SSEA-3, SSEA-4 - stage-specific embryonic antigens - as well as TRA-1-60, TRA-1-81 - keratin sulphate-associated antigens) and molecular markers (Oct-4 - POU transcriptional factor - and Nanog) (Laslett, A.L., Filipczyk, AA, and Pera, M. F. (2003) Characterization and culture of human embryonic stem cells. TCM 13., 7:295-301).
  • Oct-4 is usually the better characterized marker and the most used for the identification of mammalian pluripotent cells, being highly expressed in the inner cell mass and rapidly repressed during the differentiation process (Hansis, C 1 Grifo, J.A., Krey, L.C. (2000) Oct-4 expression in inner cell mass and trophectoderm of human blastocysts. MoI. Hum. Reprod. 6, 999- 1004) (Example 2).
  • Nanog a transcription factor recently discovered, is sufficient and necessary for the maintenance of pluripotency and is not expressed in neuronal and hematopoietic stem cells (Chambers, I., et al.
  • Nanog a pluripotency sustaining factor in embryonic stem cells.
  • Cell 113, 643-655 Ginis, I 1 et al. (2004) Differences between human and mouse embryonic stem cells, Dev. Biol. 269, 360-380; Mitsui, K. et al. (2003) The homeoprotein nanog is required for maintenance of pluripotency in mouse epiblast and ES cells, Cell 113, 631-642; Constantinescu, S. (2003) Sternness, fusion and renewal of hematopoietic and embryonic stem cells, J. Cell MoI. Med. 7, 103-112).
  • Nanog in the presence of Oct-4 has shown to be sufficient not only for the maintenance of pluripotency, but also for the stem cell self-renewal (Constantinescu, S. (2003) Sternness, fusion and renewal of hematopoietic and embryonic stem cells, J. CeII MoI. Med. 7, 103-112) (Example 3).
  • the cells obtained with the process described in the present invention are pluripotent, capable of self-renewal and continuous proliferation, of spontaneous differentiation and form homogeneous populations.
  • the cells obtained can be genetically modified, allowing several therapeutic, non-therapeutic and biotechnological applications.
  • the present invention also refers to concentrates containing stem cells, obtained from the process herein described, presenting embryonic stem cell characteristics.
  • embryonic stem cells allow the spontaneous differentiation of those cells in appropriate culture media.
  • Another object of the present invention is also an advantageous process of stem cell differentiation into several cell types, from the above described cells.
  • the surprising characteristics of said cells allow the realization of the process for differentiation in the absence of serum and/or differentiation factors, employed up to now in the processes known in the prior art. This is due to the fact that the cell differentiation in vitro generally requires the addition of various differentiation factors (for example retinoic acid, dimethyl sulfoxide, etc.), growth factors and/or cytocins.
  • various differentiation factors for example retinoic acid, dimethyl sulfoxide, etc.
  • growth factors for example retinoic acid, dimethyl sulfoxide, etc.
  • cytocins for example retinoic acid, dimethyl sulfoxide, etc.
  • the possibility of excluding the use of serum and/or inducing agents from the process, and still obtain a uniform differentiation, is especially advantageous since the serum may transmit pathogens and the differentiation factors are usually toxic and may possibly promote tumor formation in the organism.
  • the stem cells obtained as described herein may differentiate into several cell types, such as muscle, neuronal, chondrogenic, dental, hematopoietic, and endothelial.
  • the stem cells also express the CD31 marker, which is characteristic of endothelial progenitor cells (Redick, S.D., and Bautch, V.L. (1999) Developmental platelet endothelial cells adhesion molecule expression suggests multiple roles for a vascular adhesion molecule.
  • Am. J. Path. 154, 1137-1147 the opposite of endothelial precursor cells that are CD34 negative, which is similar to the profile observed in murine embryonic stem cells (Hristov, M., ErI, W., Weber, P.C. (2003) Endothelial progenitor cells. Mobilization, differentiation and homing. Aterioscler, Thromb. Vase. Biol. 23, 1185-1189).
  • the CD13 and CD31 markers in undifferentiated dental pulp cells, these may differentiate into cells of hematopoietic and endothelial types.
  • the differentiated cells obtained from the advantageous differentiation process described above are also an object of the present invention. Said cells are obtained from the process described in the present invention and present, in their undifferentiated state, a group of specific embryonic cell surface markers. Another object is the cell concentrate that comprises the differentiated cells described above.
  • the cell differentiation in a given tissue will depend on specific factors and conditions. Among these factors, the density in which cells are cultivated and the culture medium in which they are maintained are noteworthy (Example 5).
  • the cell population presented in this invention differs significantly from those previously described, since such population is homogeneous and is composed of pluripotent cells, capable of self-renewal and continuous proliferation, showing embryonic stem cell properties, able to differentiate without tumor formation.
  • the cells differentiate uniformly and easily into various cell types, allowing a broad replacement of cell populations, either damaged or deficient cells, in several tissues.
  • the stem cells of the present invention may be used in either an autologous or heterologous way in many fields of application.
  • Therapeutic and/or non-therapeutic treatment methods that include the application of cells or concentrates of the present invention are more effective than those that use the stem cells obtained through the processes known in prior art, and that do not present the said properties of embryonic stem cells and culture homogeneity.
  • therapeutic methods in accordance with the present invention those that include the application of cells and/or concentrates herein described for tissue regeneration, allowing the autologous or heterologous reconstruction; for in vitro or in vivo use of these cells in its differentiated state or not, together with scaffolds of different natures, in the presence or absence of factors that may induce their differentiation for the repair of human or animal tissues or limbs; for the use as a vehicle for carrying and introducing active molecules, proteins, peptides, oligopeptides and other molecules derived from DNA or RNA, whether modified or not (as genes in various types of different cells in vitro and ex vivo), for instance, in gene vaccine or genie and/or cell therapy.
  • said cells and concentrates that contain them are effective in the curative and/or preventive treatment of diseases such as: genetic diseases, neurodegenerative diseases, ophthalmic diseases, regeneration of cartilage and/or joints due to wear, traumas, bone regeneration due to fractures, cardiovascular diseases, angiogenic diseases, neuromuscular diseases, epidermal regeneration or replacement, among others.
  • diseases such as: genetic diseases, neurodegenerative diseases, ophthalmic diseases, regeneration of cartilage and/or joints due to wear, traumas, bone regeneration due to fractures, cardiovascular diseases, angiogenic diseases, neuromuscular diseases, epidermal regeneration or replacement, among others.
  • the stem cells with characteristics of embryonic stem cells are obtained from postnatal or adult normal tissues.
  • the present invention is also useful in the obtainment of cells from postnatal or adult tissues of patients that present pathologies or disfunctions, for example, genetic or metabolic. Said cells may then be used for the study of the etiology of pathologies, as well as in their treatment through re-implant after genetic modifications and/or transformations.
  • non-therapeutic methods of treatment that comprise the cells or concentrates of the present invention may be mentioned as examples: the use of these cells or their monolayer concentrates, in suspension or under the form of "spheres", forming embryoid bodies, a form which mimics the development of a preimplanted embryo; for the evaluation of embryo toxicity of compounds and drugs selection or identification of compounds that act on embryonic cells, since the stem cells herein described exhibit properties of embryonic stem cells; use of these cells as a support to the proliferation of undifferentiated embryonic stem cells, as well as for the production and obtainment of any type of stromal cells; for the identification of new drugs of different nature and for purposes of pharmaceutical use.
  • An additional object of the present invention includes the uses of differentiated cells of the present invention and concentrates that contain them for therapeutic, non-therapeutic and biotechnological purposes as described above.
  • the present invention refers to stem cells bank, viable due to the surprising development described above achieved by the inventors.
  • the creation and maintenance of stem cell banks are currently known in prior art. However, these banks store stem cells obtained through the processes described in prior art, and that do not show the advantageous characteristics of the stem cells obtained by the process described in the present invention. Therefore, in an additional aspect of the present invention, it is possible to store the stem cells or concentrates of the present invention in cell banks, with the objective of preserving said cells for later use. Said preservation becomes especially advantageous, due to the capacity that these cells possess to maintain their better characteristics, even after going through cryopreservation and thawing.
  • the present invention also contemplates a stem cell bank in which said cells are adult and present characteristics of embryonic stem cells, such as the cells obtained by the process described in the present invention.
  • said cell bank is directed to the preservation of stem cells obtained from dental pulp.
  • the deciduous tooth of a 5- to 6-year old child ("normal exfoliated human deciduous teeth") and the second molar of a 15-year old patient are extracted with a previous consent of the patient.
  • the dental pulp is isolated through removal of the tooth's apical part using a scalpel, followed by two washes in sterile buffered saline solution [0.01 M PBS pH 7.4] containing antibiotics [100 units/ml penicillin and 100 ⁇ g/ml streptomycin] and the transference (without any dissociation) to a 35 cm 2 Petri plate (Corning, NY) containing DMEM/F12 medium (Dulbecco's modified Eaglee's medium/Ham's
  • the dental pulp cell culture growth is maintained under these conditions for 2 weeks, and then the cells are twice washed in buffered PBS, dissociated in a 0.05% trypsin solution/EDTA (Invitrogen) and plated at a 10 4 cells/cm 2 density.
  • the culture is maintained semiconfluent aiming at preventing the differentiation of these cells. Said culture undergoes passages every 4 and 5 days with daily medium replacement.
  • the cells are resuspended in a medium containing 20% FBS, 70% DMEM and 10% dimethyl sulfoxide (DMSO, Sigma, St Louis, MO) at a 5 x 10 5 cells/ml density, the temperature being slowly and gradually decreased at a rate of 1 0 C per minute, down to the final temperature of -70 0 C. Then, after 24 hours, the cells are transferred to a tank containing liquid nitrogen.
  • a medium containing 20% FBS, 70% DMEM and 10% dimethyl sulfoxide (DMSO, Sigma, St Louis, MO) at a 5 x 10 5 cells/ml density, the temperature being slowly and gradually decreased at a rate of 1 0 C per minute, down to the final temperature of -70 0 C.
  • DMSO dimethyl sulfoxide
  • the dental pulp cell strains exhibit a morphology similar to that observed in fibroblasts, high rate of proliferation and in approximately 3 weeks, 3 x 10 7 cells are generated from each strain. After 25 passages no alterations are observed in the morphology and in the growth pattern of these cells.
  • the dental pulp cells present high capacity for clonal propagation on passage 5 and the frequency of cell colonies formation varies between 80 and 100 colonies/100 plated cells (90cm 2 ).
  • the growth kinetics of a single colony derived from dental pulp cells is measured at passage 5. During 16 days, the cells are collected and counted every day, and no change in growth rate is observed. (Figure 1A).
  • the proliferative activity of the dental pulp cells is also evaluated at different passages using the cell proliferation nuclear anti-antigen (PCNA) antibody and all cells were shown to be positive for this marker ( Figures 1B and C).
  • PCNA cell proliferation nuclear anti-antigen
  • Figures 1B and C When cultivated under n ⁇ n-adherent conditions, the dental pulp cells form spherical structures (SLS, sphere-like structure) that are composed of cells proliferatively active in the center, as evidenced by immunocytochemical analysis utilizing the antibody anti-PCNA, and by cells undergoing differentiation in the periphery ( Figures 1 D and E). These cells present a normal karyotype in all strains obtained and no alteration was observed after 10 passages.
  • SLS cell proliferation nuclear anti-antigen
  • EXPRESSION OF CELLULAR MARKERS For the immunohistochemical analysis, the cellular markers kit for embryonic stem cells (ES Cells Marker Sample Kit, Chemicon, Temecula, CA) containing the antibodies against the antigens SSEA-1 , SSEA-3, SSEA-4, (TRA)-I -60 and TRA-1-80 and Oct-4, as well as the cellular markers for mesenchymal stem cells such as SH-2, SH-3 e SH-4, is used.
  • the anti-nestin monoclonal antibodies mouse (Santa Cruz Biotechnology, CA), anti- ⁇ tubulin III and anti- ⁇ -actinin (sarcomeric) (Sigma) smooth muscle titin and anti-actin, ACSP ("aggrecan") and AFCP antibodies (Chemicon) are used.
  • the proliferative activity of the cells inside the SLS is analyzed by the utilization of PCNA together with FITC (Chemicon).
  • the following immunostaining protocol is employed: a culture of differentiated or undifferentiated dental pulp cells is grown over cover slips and twice washed with buffered Rinse (TBST, 20 mM Tris-HCI pH 7.4, 0.15 M NaCI, 0.05% Tween-20), fixed with paraformaldehyde and permeabilized with 0.1% Triton X- 100. After blocking with 5% bovine seric albumin (BSA), the cells are incubated with the diluted primary antibody for 1 hour at room temperature. The primary antibodies in the cellular markers kit are diluted 1 :40 and 1:100.
  • the fixed cells are incubated for 30 minutes, in the dark, with a mixture containing a naphtol AS-MX alkaline phosphatase solution and fast red violet salt (Lan, C.W., Wang, F.F., Wang, Y.J. (2003) "Osteogenic enrichment of bone-marrow stromal cells with the use of flow chamber and type I collagen-coated surface", J. Biomed. Mater, Res. 66A, 38-46).
  • Microscope slides are mounted with an anti-fade/DAPI or anti-fade solution.
  • the analysis are performed in a Zeiss Il epifluorescence microscope.
  • the digital images are acquired through the use of a refrigerated CCD camera (PCO, VC44), and processed with the ISIS (MetaSystem) program.
  • PCO refrigerated CCD camera
  • ISIS MetalSystem
  • a argon ion laser is used adjusted for FITC excitation at 488 nm and the emitted light is filtered through a 515 nm filter in a laser tracking microscope (LSM 410, Zeiss).
  • LSM 410 laser tracking microscope
  • the sections are taken at approximately mid height of the cell level.
  • the photomultiplier gain and the laser power are kept the same during all the experiments.
  • Embryonic stem cell surface markers such as SSEA-3 and SSEA- 4 (stage-specific embryonic antigens), Oct-4 (POU transcriptional factor), as well as TRA-1-60 and TRA-1-81 (keratin sulphate-associated antigens), are utilized for the analysis of the dental pulp cells obtained ( Figure 2). A stable expression of these markers is shown by all cell strains, both at the early (2 and 3) and the late (8 and 9) passages. The results of the dental pulp cells immunocytochemistry are summarized in Table 1. Four undifferentiated dental pulp cell strains are uniformly and strongly positive for the SSEA-4 e Oct-4 antibodies (Table 1 , Figure 2A-C), and negative for SSEA-1 (data not shown).
  • SSEA-3, TRA-1-60 and TRA-1-81 varies among the undifferentiated dental pulp cells (Table 1 , Figure 2D-F).
  • Murine embryonic stem cells and human adult skin fibroblasts are used as control (Table 1, Figure 2G-I).
  • the human dental pulp cells are also strongly positive for mesenchymal stem cells for antigen-specific, such as SH-2, SH-3 and SH-4 ( Figure 2J-M), and for alkaline phosphatase ( Figure 2N), that is usually active in both human and mice undifferentiated embryonic stem cells.
  • RNA of the cells is extracted through the use of the Trizol (Invitrogen) reagent and the single-strand cDNA is obtained using the ProStar First-Strand RT-PCR kit (Stratagene), oligo-dTi 2 and 5 ⁇ g of total RNA.
  • the following oligonucleotide primer is used for the PCR amplification: Nanog sense 5' GTCTKCTRCTGAGATGC 3', Nanog antisense 5' ASTKG I I I I I I CTGCCACC 3', PECAM/CD31 sense 5' AAGGTCAGCAGCATCGTG 3', PECAM/CD31 antisense 5'AGTGCAGATATACGTCCC 3'.
  • the PCR amplification program used was: 94°C — 3 min, 40 x (94°C-45 sec, annealing temperature specific for each primer -1 min, 72°C-1 min), 72°C-5 min.
  • the analysis by RT-PCR allow examining the Nanog expression, another marker associated with pluripotent stem cells.
  • the Nanog expression may be verified in all undifferentiated cell strains, in SLS after 3 to 8 days in culture, formed by dental pulp cells. On the other side, when these cells differentiate into smooth muscle cells, the Nanog expression is either reduced or absent (Figure 20).
  • the CD31 progenitor endothelial cell marker is expressed in undifferentiated dental pulp cells (Figure 2Q), while no GATA-4 and alpha-fetoprotein could be observed in undifferentiated dental pulp cell strains (data not shown).
  • EXAMPLE 4 FLOW CYTOMETRY ANALYSIS For the flow cytometry analysis, the antibodies against the cell surface molecules and their respective isotype controls, such as human monoclonal anti-CD45, CD13 (Sigma), CD43, CD34 (BD-Pharmigen, San Diego, CA), and CD105 (Serotec, Oxford, UK) are used. One million cells are incubated with the antibodies during 30 minutes on ice, washed in PBS containing 2% FBS and 1 ⁇ M sodium azide, followed by the addition of FITC or PE.
  • the flow cytometry analysis is performed in a FACS (Fluorescence- Activated Cell Sorter, Becton, Dickson, San Jose, CA) using the CELLQuest (Becton, Dickson) program.
  • the characterization by flow cytometry at passages 5 and 6 reveals that the dental pulp cells are positive for the CD105 marker (SH-2, mesenchymal stem cell marker), as well as for the CD13 (hematopoietic progenitor cells) ( Figure 2R, S, W), and negative for the hematopoietic cell markers CD34, CD45, CD43 ( Figure 2T-V; 1 C).
  • the cells are maintained confluent during 1 week in a 25 cm 2 culture flask, containing DMEM medium supplemented with 20% Knockout (Invitrogen) serum, 100 units/ml penicillin, 100 ⁇ g/ml streptomycin, and 2 mM L-glutamine. Then, they are harvested through the use of a 0.05% trypsin/EDTA solution and plated at high density onto 35 cm 2 Petri plates containing the same culture medium. The neuronal differentiation is also induced by the addition of all-trans retinoic acid (RA) (Sigma).
  • RA all-trans retinoic acid
  • the dental pulp cell suspension obtained by trypsinization is transferred to a 35 cm 2 Petri plate, pre-treated with a 0.1% agarose solution (Sigma), containing a neurobasal culture medium (Invitrogen) supplemented with B27. After 24 hours, the cells form spherical structures (SLS) and the neuronal differentiation is induced by the addition of RA and DMSO, to a final concentration of 10 "7 M e 0.05%, respectively, said medium being replaced every day. After 4 days of culture, under non-adhering conditions, the SLS are adhered to plates treated with 0.1% gelatin containing an appropriate culture medium.
  • a neurobasal culture medium Invitrogen
  • the undifferentiated dental pulp cells are plated at low density, /. e., 10 3 cells onto 35 cm 2 Petri plates.
  • the cells are plated at high density, /. e., 10 6 onto 35 cm 2 Petri plates, containing culture medium for undifferentiated dental pulp cells, replaced in the following day by DMEM Knockout (Invitrogen) medium supplemented with 20% Knockout serum, 100 units/ml penicillin, 100 ⁇ g/ml streptomycin, and 2 mM L-glutamine, which is utilized in the last 2 types of differentiation.
  • the same culture medium is utilized for differentiation into conjunctive tissue cells.
  • those cells are plated between 2 cover slips and the differentiation process is followed up for a long period.
  • the differentiation into skeletal muscle cells is induced by plating the cells at low density and cultivating them under serum replacement conditions (Figure 3).
  • the first myoblasts fuse together to form small myotubes (Figure 3A).
  • These immature thin and short myotubes with differentiated nucleus clustered in a central location differentiate themselves in a few days into large multinucleated syncytia ( Figure 3B).
  • contractile myofibers are formed ( Figure 3C).
  • the sarcoplasm of the majority of mature myotubes is filled with a large number of myofibrils with a striated morphology and with discernible Z-discs ( Figure 3D).
  • the differentiation for smooth muscle is induced by plating at high density in serum replacement medium.
  • the dental pulp cells under differentiation under monolayer conditions do not show inhibition by contact even after confluence ( Figure 3G), and gradually roll up like a sheet of cells resistant to trypsinization.
  • the anti-actin monoclonal antibody for smooth muscle shows a positive reaction in differentiated dental pulp cells ( Figure 3H). After confluence, the cells are capable to form multicellular spheric nodules that also express ⁇ -actinin ⁇ o
  • the SLS formed by the dental pulp cells are plated between 2 cover slips and maintained under serum replacement conditions.
  • the adherent SLS show the first signs of differentiation after 1 week of culture.
  • the chondrogeny expands from initial sites composed of undifferentiated cells that grow bigger due to the expression and accumulation of the extracellular matrix molecules rich in proteoglycans ( Figures 4A and B).
  • the cartilage formation is demonstrated through immunohistochemistry and histological staining utilizing the antibodies ACSP (anti-chondroitin sulfate proteoglycan - aggrecan) and AFCP (anti-fetal cartilage proteoglycan), and toluidine blue stain link, respectively (Figure 4C-G).
  • SLS are formed from the dental pulp cells cultivated at high density and under serum replacement conditions, for 1 week, and adhered to gelatin (Figure 5A). Three days later, the cells on top of the adhered SLS show a positive marking for nestin as well as for ⁇ -tubulin III ( Figures 5B and C), while in the periphery immunopositive cells for ⁇ -tubulin III are observed ( Figure 5D). All along the continuation of the differentiation process, the dental pulp cells present in the bottom layers of cells show positive immunomarking to GFAP, characteristic of glial cells ( Figures 5E and F), while the top layers are composed of cells positive for ⁇ -tubulin III ( Figures 5E and G).
  • the neuron nucleus migrates from the center of the cell to the periphery forming dendrites ( Figures 5H and K).
  • the neuronal differentiation of the dental pulp cells is induced through the SLS formation and by RA addition for 4 days, following the standard protocol for embryonic stem cells (Fraichard, A., Chassande, O., Bilbaut, G., Dehay, C 1 Savatier, P., Samarut, J. (1995) "In vitro differentiation of embryonic stem cells into glial cells and functional neurons". J. Cell. Sci. 108, 3181-3188), only the cells immunopositive for ⁇ -tubulin III are generated, and it is not possible to observe the formation of different layers of neuronal cells.
  • mice receive 10 6 DL3 cells delivered via intraperitoneal, intravenous or subcutaneous injection. The animals are sacrificed 1, 2 and 3 months after immunization and the tissues of interest are collected and rapidly frozen, for later verification of the presence of human cells. The DNA extraction and the PCR reactions for identification of human cells follow the conventional methods (Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) "Molecular cloning: A Laboratory Manual, 2 nd ed.”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • PCR probes used are: PCR 8a11F: ACAAATTAgCTgggTgTggC, PCR 8a11R: TAAgCTCACACTggCCCTgC; followed by A9F: CCCAgTgTTgAgCCTTTgTC, A9R:
  • AAgCCATCCgATgTAggAgA that amplifies the GBA gene in a selective way.
  • the dental pulp cells capacity to produce different tumors is also verified.
  • Nine male mice are injected with DL-3 cell strain through different routes and were observed for 3 months. No tumor could be detected in those mice.
  • the DNA of the human GBA gene is detected by PCR in several organs subjected to testing (lung, spleen, kidney, salivary gland, muscle, liver, heart and brain), 2 to 3 months after the DL-3 cell injection ( Figure 6).

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Abstract

La présente invention concerne un procédé d'obtention de cellules souches adultes qui présentent des caractéristiques de cellules souches embryonnaires, à partir de tissus postnataux et adultes ; des cellules souches adultes qui présentent des caractéristiques de cellules souches embryonnaires ; un procédé avantageux de différenciation de cellules souches et des cellules différenciées obtenues à partir de ce procédé. La présente invention fait aussi référence à de nombreuses possibilités d'application de ces cellules, qu'elles soient ou non différenciées : thérapeutiques et non thérapeutiques, biotechnologiques et pharmaceutiques, résultant du développement atteint par les inventeurs.
PCT/BR2006/000309 2006-12-29 2006-12-29 Procédé d'obtention de cellules souches WO2008080200A1 (fr)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2360434A1 (es) * 2009-07-21 2011-06-06 Universitat Internacional De Catalunya Celulas madre pluripotenciales obtenidas a partir de la pulpa dental.
WO2013011131A3 (fr) * 2011-07-20 2013-03-21 Angewandte Biotechnologie Gmbh Cellules périvasculaires pluripotentes de tendon et de ligament
AU2010271722B2 (en) * 2009-07-15 2014-05-29 Mari Dezawa Pluripotent stem cell that can be isolated from body tissue
WO2014141210A3 (fr) * 2013-03-15 2015-04-30 Avita International Ltd. Cellules souches de pulpe dentaire immatures multifonctionnelles et applications thérapeutiques
US9399758B2 (en) 2009-07-15 2016-07-26 Mari Dezawa SSEA3(+) pluripotent stem cell that can be isolated from body tissue
WO2017153957A1 (fr) * 2016-03-09 2017-09-14 Avita International Ltd. Cellules souches exprimant des marqueurs mésenchymateux et neuronaux, compositions correspondantes et procédés pour leur préparation
JP2017529068A (ja) * 2014-08-14 2017-10-05 アヴィタ・インターナショナル・リミテッド 幹細胞組成物および療法適用のための幹細胞を産生する方法
WO2019211734A1 (fr) * 2018-04-30 2019-11-07 Avita International Ltd. Cellules souches de pulpe dentaire immatures et procédés d'utilisation pour traiter une défaillance de la moelle osseuse
US11207352B2 (en) 2013-03-15 2021-12-28 Avita International Ltd. Compositions comprising stem cells expressing mesenchymal and neuronal markers and uses thereof to treat neurological disease
US11306290B2 (en) 2013-03-15 2022-04-19 Avita International Ltd. Stem cells expressing mesenchymal and neuronal markers, compositions thereof, and methods of preparation thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004094588A2 (fr) * 2003-04-19 2004-11-04 Government Of The United States Of America As Represented By The Sercretary Of The Department Of Health And Human Services National Institutes Of Health Cellules souches postnatales et utilisations
WO2005012510A1 (fr) * 2003-07-31 2005-02-10 Università Degli Studi Di Roma 'la Sapienza ' Methode permettant d'isoler et de developper des cellules souches cardiaques a partir d'une biopsie

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004094588A2 (fr) * 2003-04-19 2004-11-04 Government Of The United States Of America As Represented By The Sercretary Of The Department Of Health And Human Services National Institutes Of Health Cellules souches postnatales et utilisations
WO2005012510A1 (fr) * 2003-07-31 2005-02-10 Università Degli Studi Di Roma 'la Sapienza ' Methode permettant d'isoler et de developper des cellules souches cardiaques a partir d'une biopsie

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11261426B2 (en) 2009-07-15 2022-03-01 Mari Dezawa Pluripotent stem cell that can be isolated from body tissue
AU2010271722B2 (en) * 2009-07-15 2014-05-29 Mari Dezawa Pluripotent stem cell that can be isolated from body tissue
US9399758B2 (en) 2009-07-15 2016-07-26 Mari Dezawa SSEA3(+) pluripotent stem cell that can be isolated from body tissue
US9550975B2 (en) 2009-07-15 2017-01-24 Mari Dezawa SSEA-3 pluripotent stem cell isolated from body tissue
US9017663B2 (en) 2009-07-21 2015-04-28 Universitat Internacional De Catalunya Pluripotent stem cells obtained from dental pulp
ES2360434A1 (es) * 2009-07-21 2011-06-06 Universitat Internacional De Catalunya Celulas madre pluripotenciales obtenidas a partir de la pulpa dental.
WO2013011131A3 (fr) * 2011-07-20 2013-03-21 Angewandte Biotechnologie Gmbh Cellules périvasculaires pluripotentes de tendon et de ligament
WO2014141210A3 (fr) * 2013-03-15 2015-04-30 Avita International Ltd. Cellules souches de pulpe dentaire immatures multifonctionnelles et applications thérapeutiques
US9790468B2 (en) 2013-03-15 2017-10-17 Avita Iinternational Ltd. Multifunctional immature dental pulp stem cells and therapeutic applications
US11207352B2 (en) 2013-03-15 2021-12-28 Avita International Ltd. Compositions comprising stem cells expressing mesenchymal and neuronal markers and uses thereof to treat neurological disease
US11306290B2 (en) 2013-03-15 2022-04-19 Avita International Ltd. Stem cells expressing mesenchymal and neuronal markers, compositions thereof, and methods of preparation thereof
JP2017529068A (ja) * 2014-08-14 2017-10-05 アヴィタ・インターナショナル・リミテッド 幹細胞組成物および療法適用のための幹細胞を産生する方法
WO2017153957A1 (fr) * 2016-03-09 2017-09-14 Avita International Ltd. Cellules souches exprimant des marqueurs mésenchymateux et neuronaux, compositions correspondantes et procédés pour leur préparation
IL261623B1 (en) * 2016-03-09 2023-11-01 Avita Int Ltd Stem cells expressing mesenchymal and neuronal markers, compositions containing them and methods for their preparation
IL261623B2 (en) * 2016-03-09 2024-03-01 Avita Int Ltd Stem cells expressing mesenchymal and neuronal markers, compositions containing them and methods for their preparation
WO2019211734A1 (fr) * 2018-04-30 2019-11-07 Avita International Ltd. Cellules souches de pulpe dentaire immatures et procédés d'utilisation pour traiter une défaillance de la moelle osseuse

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