WO2010057965A1 - Cellules souches mésenchymateuses d’origine myométriale - Google Patents

Cellules souches mésenchymateuses d’origine myométriale Download PDF

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Publication number
WO2010057965A1
WO2010057965A1 PCT/EP2009/065523 EP2009065523W WO2010057965A1 WO 2010057965 A1 WO2010057965 A1 WO 2010057965A1 EP 2009065523 W EP2009065523 W EP 2009065523W WO 2010057965 A1 WO2010057965 A1 WO 2010057965A1
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Prior art keywords
cells
cell population
tissue
stem cell
cell
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PCT/EP2009/065523
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English (en)
Inventor
Beatriz GONZÁLEZ GÁLVEZ
Juan Carlos RODRÍGUEZ CIMADEVILLA
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Projech Science To Technology, S.L.
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Priority to JP2011536872A priority Critical patent/JP2012509074A/ja
Priority to AU2009317191A priority patent/AU2009317191A1/en
Priority to CA2744423A priority patent/CA2744423A1/fr
Priority to BRPI0921414A priority patent/BRPI0921414A2/pt
Priority to EP09752860A priority patent/EP2366023A1/fr
Publication of WO2010057965A1 publication Critical patent/WO2010057965A1/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
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
    • C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells

Definitions

  • stem cell therapy holds tremendous promise for repair and/or regeneration of aging and damaged tissue.
  • ES cells embryonic stem cells
  • adult stem cells Depending on the origin of the stem cells, we can differentiate between embryonic stem cells (ES cells) and adult stem cells.
  • the ES cells come from the internal cellular mass of the blastocyte and their most relevant feature is the fact that they are pluripotential, which means that they can give rise to any adult tissue derived from the three embryonic layers.
  • ES cells are partially compromised cells present in adult tissue which can remain in the human body for decades although they become scarcer with the passage of time.
  • Document EP 1876233 describes the isolation of a cell population which originate in an endometrial tissue or from an endometrial tissue isolated from a menstrual blood, a cord blood or an appendage of a fetus. These cells can differentiate into cardiac muscle cells.
  • Masanori O. et al (PNAS, 2007. vol. 104, 47: 18700-18705) have described the isolation of a side population in human uterine myometrium with phenotypic and functional characteristics of stem cells. Said cells are positive for the surface markers CD90, CD73, CD105, CD34 and STRO-I and negative for CD44. Said cells were able to differentiate into adipocyte, osteocyte and smooth muscle cells.
  • the isolation of said cell population is carried out by means of hysterectomy, i.e. by surgical removal of the uterus.
  • the invention refers to a pharmaceutical composition
  • a pharmaceutical composition comprising an isolated stem cell population according to the invention and an acceptable pharmaceutical vehicle.
  • the invention refers to a method for isolating a stem cell population from myometrial tissue, wherein the cells of said cell population are characterized in that are positive for CD31, CD34, CD44, CDl 17, SSEA-4 and WGA- lectin surface markers and negative for CD13, CD45, CD80, CD133, CD146, TRAl-60 and TRAl -81 surface markers, said method comprising the steps of: i) incubating a myometrial tissue sample in a suitable cell culture medium on a solid surface under conditions allowing cells of said sample to adhere to said solid surface; ii) recovering the cells from said cell culture which do not adhere to said solid surface or which present low adherence capacity; and iii) confirm that the selected cell population presents the phenotype of interest.
  • Figure 1 Surface markers expression analyzed by flow cytometry. FACS analysis using a panel of antibodies: CD13, CD31, CD34, CD44, CD45, CD80, CD90, CDl 17,
  • Figure 4 EMSCs immuno staining for nestin after one week in culture in neural stem cell proliferation medium. Phase contrast (left panel), Nestin (middle panel), nuclei (right panel). (Microphoto graphs taken with a Nikon camera in a Nikon Fluorescence Inverted Microscope with a 2Ox objective).
  • the present invention refers to a new mesenchymal stem cell population which has been isolated from myometrial tissue, said stem cell population showing capability to differentiate into multiple cell types in vitro.
  • the present invention refers to an isolated, myometrial- derived mesenchymal stem cell population, hereinafter referred to as "cell population of the invention", characterized in that the cells of said cell population are positive for CD31, CD34, CD44, CDl 17, SSEA-4 (Stage-specific embryonic antigen-4), HLA-DR and WGA-lectin (wheat germ agglutinin- lectin) surface markers and negative for CD 13, CD45, CD80, CD133, CD146, TRAl-60 and TRA1-81 (Tumor Rejection Antigen 1) surface markers.
  • MHC major histocompatibility complex
  • HLA human leukocyte antigen
  • the term "isolated” applied to a cell population refers to a cell population, isolated from the human or animal body, which is substantially free of one or more cell populations that are associated with said cell population in vivo or in vitro.
  • the cells of the cell population of the invention hereinafter referred to as the "cells of the invention", derive from the myometrial tissue.
  • myometrial tissue refers to tissue derived from the middle layer of the uterine wall.
  • uterus as used herein, encompasses the cervical canal and uterine cavity.
  • the term “uterine tissue” refers to any material in the cervical canal and uterine cavity.
  • the cells of the invention can be obtained from any suitable source of myometrial tissue from any suitable animal, including humans. In general, said cells are obtained from non-pathological post-natal mammalian myometrial tissue. In a particular embodiment, the cells of the cell population of the invention are from a mammal, e.g , a rodent, primate, etc, preferably, from a human.
  • the cells of the invention are characterized in that are positive for CD31, CD34, CD44, CDl 17, SSEA-4, HLA-DR and WGA-lectin surface markers and negative for CD13, CD45, CD80, CD133, CD146, TRAl-60 and TRAl -81 surface markers.
  • the expression "significant expression” means that, in a cell population comprising the cells of the invention, more than 10%, preferably 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or all of the cells show a signal for a specific cell surface marker by flow cytometry above the background signal using conventional methods and apparatus (for example a Calibur (Becton Dickinson) FACS system used with commercially available antibodies and standard protocols known in the art).
  • a Calibur Becton Dickinson
  • the term "gene” as used herein, may be a gene comprising transcriptional and/or translational regulatory sequences and/or a coding region and/or non- translated sequences (e.g., introns, 5'- and 3 '-untranslated sequences).
  • the coding region of a gene may be a nucleotide sequence coding for an amino acid sequence or a functional RNA, such as tRNA, rRNA, catalytic RNA, siRNA, miRNA or antisense RNA.
  • a gene may also be an mRNA or cDNA corresponding to the coding regions (e.g., exons and miRNA) optionally comprising 5'- or 3 '-untranslated sequences linked thereto.
  • a gene may also be an amplified nucleic acid molecule produced in vitro comprising all or a part of the coding region and/or 5'- or 3 '-untranslated sequences linked thereto.
  • gene expression refers to a process that involves transcription of the DNA code into mRNA, translocation of mRNA to ribosomes, and translation of the RNA message into proteins.
  • the determination of the expression levels of said genes can be carried out by any standard method known in the state of the art.
  • said methods include measuring the expression levels of the mRNA encoded by the above mentioned genes.
  • a biological sample comprising the cells of the invention may be treated to physically or mechanically disrupt cell structure, to release intracellular components into an aqueous or organic solution to prepare nucleic acids for further analysis.
  • the nucleic acids are extracted from the sample by procedures known to the skilled person and commercially available.
  • RNA is then extracted by any of the methods typical in the art, for example, Sambrook, Fischer and Maniatis, Molecular Cloning, a laboratory manual, (2nd ed.), Cold Spring Harbor Laboratory Press, New York, (1989). Preferably, care is taken to avoid degradation of the RNA during the extraction process. While all techniques of gene expression profiling are suitable for use in performing the foregoing aspects of the invention, the gene mRNA expression levels are often determined by reverse transcription polymerase chain reaction (RT-PCR).
  • RT-PCR reverse transcription polymerase chain reaction
  • the detection method provides an output (i.e., readout or signal) with information concerning the presence, absence of the marker(s) in a sample.
  • the output may be qualitative (e.g., "positive” or “negative”).
  • "positive gene expression” is considered when an amplification product of said gene using any standard amplification reaction is obtained.
  • Means for evaluating or detecting said amplification product are well known in the state of the art. In an illustrative way, said methods include, for example, visualisation of a band in an agarose gel as shown in the Example 1 accompanying the present invention. In order to carry out said amplification reaction, specific amplification oligonucleotides for said genes are used.
  • the cells of the cell population of the invention present capacity to be differentiated into smooth muscle cells.
  • the cells of said population present capacity to be differentiated into adipocytes.
  • the cells of said population present capacity to be differentiated into osteoblasts.
  • the cells of said population present capacity to be differentiated into neural cells.
  • the cells of the invention are also capable of being expanded ex vivo. That is, after isolation, the cells of the invention can be maintained and allowed to proliferate ex vivo in culture medium.
  • culture medium is composed of, for example, Dulbecco's Modified Eagle's Medium (DMEM), with antibiotics (for example, 100units/ml Penicillin and 100 ⁇ g/ml Streptomycin) or without antibiotics, and 5 mM glutamine, and supplemented with 2-20% fetal bovine serum (FBS). It is within the skill of one in the art to modify or modulate concentrations of media and/or media supplements as necessary for the cells used. Sera often contain cellular and non-cellular factors and components that are necessary for viability and expansion.
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS fetal bovine serum
  • Antimicrobial agents are also typically used in cell culture to mitigate bacterial, mycoplasmal, and fungal contamination.
  • antibiotics or anti-mycotic compounds used are mixtures of penicillin/streptomycin, but can also include, but are not limited to amphotericin (Fungizone(R)), ampicillin, gentamicin, bleomycin, hygromacin, kanamycin, mitomycin, etc.
  • the maintenance conditions of the cells of the invention can also contain cellular factors that allow cells to remain in an undifferentiated form. It is apparent to those skilled in the art that prior to differentiation, supplements that inhibit cell differentiation must be removed from the culture medium. It is also apparent that not all cells will require these factors. In fact, these factors may elicit unwanted effects, depending on the cell type.
  • a cell is said to be "genetically modified", “transfected”, or “genetically transformed” when a polynucleotide has been transferred into the cell by any suitable means of artificial manipulation, or where the cell is a progeny of the originally altered cell that has inherited the polynucleotide.
  • the polynucleotide will often comprise a transcribable sequence encoding a protein of interest, which enables the cell to express the protein at an elevated level.
  • the genetic alteration is said to be “inheritable” if progeny of the altered cell have the same alteration.
  • amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but which functions in a manner similar to a naturally occurring amino acid.
  • MSC have been increasingly given a role in tissue repair and regeneration. In different models of tissue damage, MSC improve the recovery of injured tissues.
  • the cells of the invention express some of the proteins that leukocytes use to adhere to and cross the endothelium (CD44) and thus can diffuse into the interstitium of the skeletal muscle, inside the osteogenic tissue and between the adypocytes, when delivered intra-arterially.
  • CD44 endothelium
  • the data presented herein demonstrate that the cells of the invention can be grown extensively but not indefinitely in vitro, which is of vital importance considering that an additional concern for future cell therapy protocols is the risk that extensive expansion in vitro may compromise differentiation and/or self-renewal ability or even lead to malignant transformation. Indeed, as mentioned above, said cells undergo senescence after approximately 30 passages in vitro.
  • karyotype refers to the chromosome characteristics of an individual cell or cell line of a given species, as defined by both the number and morphology of the chromosomes.
  • the karyotype is presented as a systematized array of prophase or metaphase (or otherwise condensed) chromosomes from a photomicrograph or computer-generated image.
  • interphase chromosomes may be examined as histone-depleted DNA fibres released from interphase cell nuclei. It is considered a normal karyotype when the number of chromosomes is not altered compared to the number of chromosomes of the specie.
  • treat refers to the amelioration of one or more symptoms associated with a disorder that results from the administration of the cell population of the invention or a pharmaceutical composition comprising same, to a subject in need of said treatment.
  • subject refers to an animal, preferably a mammal including a non- primate (e.g. a cow, pig, horse, cat, dog, rat, or mouse) and a primate (e.g. a monkey or a human). In a preferred embodiment, the subject is a human.
  • a non- primate e.g. a cow, pig, horse, cat, dog, rat, or mouse
  • a primate e.g. a monkey or a human.
  • the subject is a human.
  • carrier in the context of the present invention denotes any one of inert, non-toxic materials, which do not react with the cell population of the invention and which can be added to formulations as diluents, adjuvants, excipients, or vehicle or to give form or consistency to the formulation.
  • the carrier may at times have the effect of the improving the delivery or penetration of the active ingredient to the target tissue, for reducing undesired side effects etc.
  • the carrier may also be a substance that stabilizes the formulation (e.g. a preservative), for providing the formulation with an edible flavor, etc.
  • stabilizers and adjuvants see E. W.
  • compositions can also contain minor amounts of pH buffering agents.
  • Such compositions will contain a prophylactic or therapeutically effective amount of a prophylactic or therapeutic agent preferably in purified form, together with a suitable amount of earner so as to provide the form for proper administration to the subject.
  • the formulation should suit the mode of administration.
  • the pharmaceutical compositions are sterile and in suitable form for administration to a subject, preferably an animal subject, more preferably a mammalian subject, and most preferably a human subject.
  • the active materials can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, or have another action.
  • the compounds, i.e. the cell population of the invention may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients, such as, for example other agents useful in the treatment of a tissue degenerative condition.
  • the cell population and composition of the invention may be administered in a combination therapy.
  • combination therapy refers to the use of the cell populations of the present invention with other active agents or treatment modalities, in the manner of the present invention for the amelioration of one or more symptoms associated with a disorder. These other agents or treatments may include known drugs and therapies for the treatment of such disorders.
  • the combined use of the agents of the present invention with other therapies or treatment modalities may be concurrent, or given sequentially, that is, the two treatments may be divided up such that a cell population or a pharmaceutical composition comprising same of the present invention may be given prior to or after the other therapy or treatment modality.
  • the attending physician may decide on the appropriate sequence of administering the cell population, or a pharmaceutical composition comprising same, in combination with other agents, therapy or treatment modality.
  • the present invention relates to the use of the cells of the invention for the preparation of a medicament for preventing, treating or ameliorating one or more symptoms associated with a tissue degenerative condition including, but not limited to, skeletal muscle degeneration, cardiac tissue degeneration, bone tissue degeneration, neural tissue degeneration, lung degeneration, liver degeneration, kidney degeneration or more than one of said tissue degenerative conditions simultaneously.
  • a tissue degenerative condition including, but not limited to, skeletal muscle degeneration, cardiac tissue degeneration, bone tissue degeneration, neural tissue degeneration, lung degeneration, liver degeneration, kidney degeneration or more than one of said tissue degenerative conditions simultaneously.
  • the present invention refers to a method, hereinafter referred to as the "method of the invention", for isolating a stem cell population from myometrial tissue, wherein the cells of said cell population are characterized in that are positive for CD31, CD34, CD44, CDl 17, SSEA-4 and WGA-lectin surface markers and negative for CD13, CD45, CD80, CD133, CD146, TRA1-60 and TRAl -81 surface markers, said method comprising the steps of: i) incubating a myometrial tissue sample in a suitable cell culture medium on a solid surface under conditions allowing cells of said sample to adhere to said solid surface; ii) recovering the cells from said cell culture which do not adhere to said solid surface or which present low adherence capacity; and iii) confirm that the selected cell population presents the phenotype of interest.
  • solid surface refers to any material that allows cells to adhere.
  • said material is gelatin.
  • myometrial tissue samples were transferred to a Petri dish coated with gelatin 1% as previously described for other cell types (Minasi, M.G., et al cited supra; Sampaolesi M, et al. 2003. Science. 301 :487-492). These samples were cultured for 15 days and after the initial outgrowth of fibroblast-like cells, small round and refractile cells appeared. . Those cells, which adhered poorly to the substratum and floated, were easily collected by gently pipetting from the original culture.
  • the expression "low adherence capacity" as used herein refers to cells which, under standard conditions allowing cells (such as, for example, fibroblast) adhere to said solid surface, either do not adhere to said solid surface and thus, float in the culture medium, or can easily be collected from said culture medium by means, for example, of gently pipetting.
  • cells are cultured without differentiation on a solid surface, usually made of gelatin in the presence of a suitable cell culture medium [e g , DMEM, typically supplemented with 5-15% (e g , 10%) of a suitable serum, such as fetal bovine serum or human serum], and incubated under conditions which allow cells to adhere to the solid surface and proliferate.
  • a suitable cell culture medium e g , DMEM, typically supplemented with 5-15% (e g , 10%) of a suitable serum, such as fetal bovine serum or human serum
  • the cells are maintained in culture in the same medium and under the same conditions until they reach the adequate confluence, typically, about 80% cell confluence, with replacement of the cell culture medium when necessary.
  • the cells can be expanded by means of consecutive passages using a detachment agent such as trypsin and seeding onto a bigger cell culture surface at the appropriate cell density (usually 2,000- 10,000 cells/cm 2 )
  • a detachment agent such as trypsin
  • seeding onto a bigger cell culture surface at the appropriate cell density (usually 2,000- 10,000 cells/cm 2 )
  • the cells are then passaged at least twice in such medium without differentiating, while still retaining their developmental phenotype, and more preferably, the cells can be passaged at least 10 times (e g , at least 15 times or even at least 20 times) without losing developmental phenotype
  • the cells are plated at a desired density such as between about 100 cells/cm2 to about 100,000 cells/cm2 (such as about 500 cells/cm2 to about
  • Example 1 describes in a detailed manner the isolation of the cells of the invention from mouse myometrial tissue.
  • Cell-surface markers can be identified by any suitable conventional technique, usually based on a positive/negative selection, for example, monoclonal antibodies against cell-surface markers, whose presence/absence in the cells has to be confirmed, can be used, although other techniques can also be used.
  • monoclonal antibodies against one, two, three, four, five, six, seven of or preferably all of CD13, CD45, CD80, CD133, CD146, TRAl-60 and TRAl -81 surface markers are used in order to confirm the absence of said markers in the selected cells, and monoclonal antibodies against one, two, three, four, of or preferably all of CD31, CD34, CD44, CDl 17, SSEA-4, HLA-DR and WGA-lectin are used in order to confirm the presence thereof or detectable expression levels of, at least one of and preferably all of, said markers.
  • Said monoclonal antibodies are known, commercially available or can be obtained by a skilled person in the art by conventional methods.
  • the cells and cell populations provided by the instant invention can be clonally expanded, if desired, using a suitable method for cloning cell populations.
  • a proliferated population of cells can be physically picked and seeded into a separate plate (or the well of a multi-well plate).
  • the cells can be subcloned onto a multi- well plate at a statistical ratio for facilitating placing a single cell into each well (e.g., from about 0,1 to about 1 cell/well or even about 0,25 to about 0,5 cells/well, such as 0,5 cells/well).
  • the cells can be cloned by plating them at low density (e.g., in a Petri dish or other suitable substrate) and isolating them from other cells using devices such as a cloning rings.
  • the production of a clonal population can be expanded in any suitable culture medium.
  • the isolated cells can be cultured to a suitable point when their developmental phenotype can be assessed.
  • any of the steps and procedures for isolating the cells of the cell population of the invention can be performed manually, if desired.
  • the process of isolating such cells can be facilitated and/or automated through one or more suitable devices, examples of which are known in the art.
  • the invention refers to a kit comprising a cell population containing the cells of the invention.
  • the present invention refers to the use of a cell population containing cells of the invention for preventing, treating, or ameliorating one or more symptoms associated with a tissue degenerative condition in a subject suffering from said disorders or diseases.
  • the present invention provides methods of preventing, treating, or ameliorating one or more symptoms associated with a tissue degenerative condition, in a subject suffering from said disorders or diseases, which comprises administering to said subject in need of such treatment of a prophylactically or therapeutically effective amount of a cell population containing cells of the invention.
  • said tissue degenerative condition is skeletal muscle degeneration, cardiac tissue degeneration, bone tissue degeneration, neural tissue degeneration, lung degeneration, liver degeneration, kidney degeneration or more than one of said tissue degenerative conditions simultaneously.
  • Myometrial explants were taken from the lower uterine segment of the corpus uteri by uterine exfolation. Sampling involves collecting exfoliated cells from the endocervical uterine canal with an appropiate tool. All explants were trimmed of endometrial, serosal, fat and fibrous tissue prior to use. Technique would be similar to a cervical cytology.
  • Myometrial tissue can be maintained for up to 24 hours post-obtention in oxygenated (95% O 2 , 5% CO 2 ) physiological salt solution (PBS) at room temperature. Samples were transferred to a Petri dish coated with gelatin 1% in presence of 10% FBS-DMEM plus 5 mM glutamine and antibiotics. These samples were cultured for 15 days and after the initial outgrowth of f ⁇ broblast-like cells, small round and refractile cells appeared. This cell population was easily collected by gently pipeting of the original culture, counted and cloned by limited dilution on gelatin 1% coated p96-well dishes. Myometrial precursors were also obtained from uterine explants.
  • PBS physiological salt solution
  • Myometrial tissue pieces (10-30mg) obtained from 4 months C57 mice were kept in DMEM w/o FCS (fetal calf serum), with antibiotics. Each piece was then rinsed in PBS with Ca/Mg and sharply dissected into 1-2 mm diameter pieces with a scalpel. Fragments containing small vessels were transferred to a Petri dish coated with gelatin 1% in presence of 10% FBS-DMEM plus 5 mM glutamine and antibiotics. These fragments were cultured for 15 days and after the initial outgrowth of f ⁇ broblast-like cells, small round and refractile cells appeared. This cell population was easily collected by gently pipeting of the original culture, counted and cloned by limited dilution on gelatin 1% coated p96well dishes.
  • DMEM w/o FCS fetal calf serum
  • Differentiation into neural cells consist on changing the culture medium to a neural stem cell proliferation medium: DMEM:F12 medium (Sigma) supplemented with D-Glucose (Sigma) to a final concentration of 4.5 mg/ml, N2 Supplement (Gibco- Invitrogen), B27 Supplement (Gibco-Invitrogen), 20 ⁇ g/ml insulin (Sigma), 2 ⁇ g/ml heparin (Sigma), 20 ng/ml ⁇ FGF (Sigma), 10 ng/ml EGF (Sigma).
  • the neural stem cell proliferation medium was changed twice and after one week in culture, the cells were processed for immuno cytochemistry.
  • Cells were plated at a density of 3 x 10 3 cells/cm 2 in different media and passed on average every three days. At each passage, the number of cells was counted in triplicate in a hemocytometer. For the growing curve of the clones, cells were plated initially at 1 x 10 4 cells/cm 2 in complete DMEM or embryonic media and passed every five days. At each passage, the number of cells was counted in triplicate in the hemocytometer.
  • mice were injected subcutaneously with 10 7 MAMps. After 4 months, the mice were sacrificed and analyzed for the presence of macro scopically detectable tumors.
  • Cells were grown on gelatin coated glass coverslips, washed with PBS and fixed with 4% paraformaldehyde for 10 minutes. Samples were frozen in liquid nitrogen cooled isopentane and serial 8 ⁇ m thick sections were cut with a Leyca cryostat. Cells were permeabilized with 0.2% Triton X-100, 1% BSA in PBS for 30 minutes at RT, while tissue sections were incubated without detergent. Cells and tissue sections were incubated with 10% donkey serum for 30 minutes a RT, and incubated overnight at 4°C with primary antibodies at the appropriate dilution.
  • anti-laminin monoclonal or polyclonal antibodies (Sigma) at 1 :100 dilution; MF20 antibody at 1 : 5 dilution, anti Smooth Alpha actin 1 :300 dilution from Sigma, polyclonal anti-nestin antibody (Abeam), beta-III- tubulin (anti- TUJl antibody, Abeam), doublecortin (anti- Dcx antibody, Abeam), and MAP2 (Sigma) as neuronal marker, GFAP (Sigma) as astrocyte marker and RIP (Developmental Studies Hybridoma Bank) as oligodendrocyte marker. Nuclei were stained with bisbenzimide (Sigma).
  • the conditions for the PCR were general for all primers: 94 0 C for 4 minutes. 30 cycles of 94 0 C, 45 s; 55 0 C, 45 s; 72 0 C, 45 s. And a final step of 72 0 C for 10 minutes.
  • Mef2a primer forward TTGAGGCTCTGAACAAGAAGG Mef2a primer reverse: GCATTGCCAGTACTTGGTGG MefZc primer forward: AACACGGGGACTATGGGGAGAAA MefZc primer reverse: TATGGCTGGACACTGGGATGGTA Tbx2 primer forward: GGTGCAGACAGACAGTGCGT Tbx2 primer reverse: AGGCCAGTAGGTGACCCATG Tbx5 primer forward: CCAGCTCGGCGAAGGGATGTTT Tbx5 primer reverse: CCGACGCCGTGTACCGAGTGAT Sox2 primer forward: GGCAGCTACAGCATGATGCAGGAGC Sox2 primer reverse: CTGGTCATGGAGTTGTACTGCAGG mTERT: human/mouse TERT primer pair (R&D systems)
  • Stain solution was added (mix fast red violet with naphthol, phosphatase solution and water in a 2:1 :1 ratio. Detection kit, (Millipore) covering each well and incubated for 15 min at room temperature in dark. Aspirate solution, rinse plates with PBS Ix and count and analyze under microscope the number of violet cells.
  • uterus biopsies were dissected under the microscope; fragments of vessels and surrounding mesenchymal tissue were dissected and plated on gelatin coated dish as previously described for other cell types (Minasi, M. G., et al cited supra; Sampaolesi M, et al. 2003. cited supra). After the initial outgrowth of f ⁇ broblast- like cells, small round and refractile cells appeared. Those cells adhered poorly to the substratum and were thus collected by gently pipetting. Floating cells were either grown as a polyclonal population or, in some cases, cloned by limited dilution.
  • MAMps clones were analyzed by flow cytometry for the expression at the cell surface of the following stem cells markers: CD31, CD34, CD44, CDl 17, alkaline phosphatase (PAL), HLA-DR, SSEA-I, HLA-DR, WGA, CD13, CD45, CD80, CD90, CD133, CD146, TRA1-60/81 and Tetramethyl Rhodamine Methyl Ester (TMRM). All clones were positive for CD31, CD34, CD44, CDl 17, SSEA-4, HLA-DR and WGA- lectin surface markers and negative for CD13, CD45, CD80, CD133, CD146, TRA1-60 and TRAl -81 surface markers. See Table 1 below for percentage and Figure 1 for FACS profiles.
  • Table 1 Percentage of expression of markers at the surface of MAMps.
  • MAMps were positive for MefZc, Sox2, Tbx5 and hTERT, while negative for MefZa and Tbx2 (see
  • MAMps readily differentiate into smooth muscle, adipocytes or osteoblasts, when treated with transforming growth factor beta (TGF ⁇ ), insulin-dexamethazone or bone morphogenetic protein 2 (BMP2).
  • TGF ⁇ transforming growth factor beta
  • BMP2 bone morphogenetic protein 2
  • the present invention shows the isolation of myometrial precursors from mouse adult uterine tissue. Said precursors can grow until 30 passages and express stem cells surface markers and genes. Besides, these precursors are able to differentiate into different mesoderm tissues types which could make them suitable for regenerative medicine. Myometrial precursors can be easily isolated from the very biopsy that is used for diagnosis, with no need of additional surgical intervention. The source of cells is important not only for practical reasons. Multipotent mesoderm progenitors, receive some sort of local commitment that favours recruitment into the cell types of the tissue where they reside. So, it is interesting to have a source of mesoderm progenitors that still remain with the multipotency property.
  • EPC endothelial progenitor cells
  • MPC multipotent adult progenitor cells
  • SP side population cells
  • mesoangioblasts stem/progenitor cells from muscle endothelium, sinovia, dermis, and adipose tissue.
  • EPC endothelial progenitor cells
  • MPC multipotent adult progenitor cells
  • SP side population cells
  • mesoangioblasts mesoangioblasts
  • the myometrial precursors of the invention express some of the proteins that leukocytes use to adhere to and cross the endothelium and thus can diffuse into the interstitium of the skeletal muscle, inside the osteogenic tissue and between the adypocytes, when delivered intra-arterially.

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Abstract

La présente invention concerne des procédés d’isolement de cellules souches adultes, les cellules ainsi isolées et des applications de celles-ci. Plus spécifiquement, l’invention concerne des cellules souches adultes isolées qui sont dérivées du myomètre, qui peuvent être différenciées en de nombreux types de tissu de mésoderme différents, comprenant le muscle lisse, les adipocytes, les ostéoblastes, les muscles squelettiques et le tissu neural, de manière à les rendre adaptées pour la médecine régénératrice.
PCT/EP2009/065523 2008-11-20 2009-11-20 Cellules souches mésenchymateuses d’origine myométriale WO2010057965A1 (fr)

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JP2011536872A JP2012509074A (ja) 2008-11-20 2009-11-20 子宮筋層由来間葉系幹細胞
AU2009317191A AU2009317191A1 (en) 2008-11-20 2009-11-20 Myometrial-derived mesenchymal stem cells
CA2744423A CA2744423A1 (fr) 2008-11-20 2009-11-20 Cellules souches mesenchymateuses d'origine myometriale
BRPI0921414A BRPI0921414A2 (pt) 2008-11-20 2009-11-20 população de células-tronco mesenquimais isoladas, composição farmacêutica, e, método para isolar uma população de células-tronco de tecido miometrial.
EP09752860A EP2366023A1 (fr) 2008-11-20 2009-11-20 Cellules souches mésenchymateuses d origine myométriale

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EP08380324.7 2008-11-20

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WO2011042547A1 (fr) * 2009-10-08 2011-04-14 Projech Science To Technology, S.L. Cellules souches mésenchymateuses d'origine myométriale et leurs utilisations
JP2013536860A (ja) * 2010-08-31 2013-09-26 クック・ジェネラル・バイオテクノロジー・エルエルシー 動物の疾病の治療のための全身的、同種間幹細胞治療

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JP2017529101A (ja) * 2014-09-18 2017-10-05 ナショナル ヘルス リサーチ インスティテュートス 前駆細胞、それらの調製方法およびそれらの使用

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WO2008148105A1 (fr) * 2007-05-25 2008-12-04 Medistem Laboratories, Inc. Cellules souches endométriales et procédés de génération et d'utilisation de celles-ci

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011042547A1 (fr) * 2009-10-08 2011-04-14 Projech Science To Technology, S.L. Cellules souches mésenchymateuses d'origine myométriale et leurs utilisations
JP2013536860A (ja) * 2010-08-31 2013-09-26 クック・ジェネラル・バイオテクノロジー・エルエルシー 動物の疾病の治療のための全身的、同種間幹細胞治療

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AU2009317191A1 (en) 2011-06-30
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CA2744423A1 (fr) 2010-05-27
BRPI0921414A2 (pt) 2019-09-24

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