WO2015080376A1 - Procédé pour différencier des cellules nerveuses et des cellules ciliées et des cellules souches mésenchymateuses dérivées du chorion placentaire ou de la gelée de wharton - Google Patents

Procédé pour différencier des cellules nerveuses et des cellules ciliées et des cellules souches mésenchymateuses dérivées du chorion placentaire ou de la gelée de wharton Download PDF

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WO2015080376A1
WO2015080376A1 PCT/KR2014/009485 KR2014009485W WO2015080376A1 WO 2015080376 A1 WO2015080376 A1 WO 2015080376A1 KR 2014009485 W KR2014009485 W KR 2014009485W WO 2015080376 A1 WO2015080376 A1 WO 2015080376A1
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cells
derived
stem cells
growth factor
hair
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박경호
길기철
최미영
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가톨릭대학교 산학협력단
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    • 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
    • C12N5/069—Vascular Endothelial cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
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    • 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
    • C12N5/0618—Cells of the nervous system
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    • 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
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0627—Hair cells
    • C12N5/0628—Hair stem cells; Hair progenitors
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    • 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
    • C12N5/066—Tenocytes; Tendons, Ligaments
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
    • C12N2506/025—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells from extra-embryonic cells, e.g. trophoblast, placenta

Definitions

  • the present invention relates to a method for differentiating stem cells isolated from chorion or warton's jelly of human placenta, and more particularly, to stems separated from chorion or wort umbilical cord of human placenta.
  • the present invention relates to a method of differentiating a chorionic or wort-collage-derived stem cell into neural cell precursors, hair cells, and nerve cells, which comprises culturing the cells in a medium containing nerve growth factors.
  • the present invention also relates to a composition for the prevention or treatment of hearing loss comprising the differentiated neuronal cell precursors, hair cells, neurons as an active ingredient.
  • organ transplantation and gene therapy have been suggested for the treatment of intractable disease in humans, but effective practical use has been insufficient due to immunorejection and lack of supply organs, vector development or lack of knowledge of disease genes.
  • stem cell research As interest in stem cell research increased, pluripotent stem cells with the ability to form all organs through proliferation and differentiation were found to be able to fundamentally solve long-term damage as well as most diseases. In addition, many scientists have suggested the possibility of applying stem cells to treatment of almost all organs of the human body as well as treatment of Parkinson's disease, various cancers, diabetes and spinal cord injury.
  • stem cell which supplies cells from the outside when cells are destroyed or damaged by disease, has been suggested as an effective treatment, especially stem cells capable of proliferation and differentiation into tissues requiring regeneration. (stem cell) is in the spotlight.
  • Neural stem cells with such diverse functions are the cells before the stage of differentiation into each cell constituting the tissue, capable of infinite proliferation in the undifferentiated state and having the potential to differentiate into cells of various tissues by specific differentiation stimulation.
  • Neural stem cells also have the ability to self-replicate and can differentiate into neurons or glia, such as astrocytes, oligodendrocytes or Schwann cells. It is an undifferentiated cell with differentiation ability, and neural stem cells are differentiated into neural cells, for example, neurons or glia, through the steps of neural progenitor cells or glia progenitor cells that produce specific nervous system cells.
  • mesenchymal stem cells are differentiated into bone, cartilage, adipose tissue, muscle, tendons, ligaments, and neural tissues, and thus are attracting attention as cells suitable for cell therapy.
  • bone marrow is the most representative tissue from which mesenchymal stem cells can be obtained.
  • mesenchymal stem cells present in bone marrow have limited application range due to their limited differentiation and proliferative capacity. Due to limitations derived from bone marrow, several stages of procedure are required, and the procedure is complicated. It is usually accompanied by mental and physical suffering.
  • a donor does not show a graft-versus-host response because the antigenic phenotypes are matched through histocompatibility antigen comparison.
  • mesenchymal stem cells were isolated from cord blood as a source of fetal mesenchymal stem cells (fetal MSC), the number was very small and there was a problem that proliferation was not good. Therefore, it is necessary to develop a new method for separating and culturing stem cells that are easily proliferated and capable of mass cultivation as cell therapeutics.
  • the loss of hearing which is one of the human senses, causes personal disturbances in daily life and enormous losses in socio-economics. Hearing loss does not only cause hearing impairment, but if severe hearing loss occurs before language acquisition, it becomes a problem due to language impairment due to normal language development.
  • the senile deafness is one of the three major senile diseases together with hypertension and degenerative arthritis. Most of the senile hearing loss are known as sensory and neural types caused by degenerative changes in auditory hair cells and neuronal ganglions.
  • the epithelial growth factor may be 5 to 15 ng / ml in the medium, and the fibroblast growth factor may be included in the medium 15 to 25 ng / ml.
  • the neuronal growth factor is at least one selected from the group consisting of glioblastoma-derived neuronal growth factor, brain-derived neurotrophic factor and neurotrophin-3, and the chorion or wharton umbilical cord-derived stem cells as hair cells or neurons. It may be to differentiate.
  • the prevention or treatment of hearing loss comprising one selected from the group consisting of neuronal cell precursors, hair cells and neurons differentiated from the chorion membrane derived from the chorionic membrane or Wharton preparations of human placenta as an active ingredient It relates to a composition for.
  • the hair cells or the neurons may comprise one or more nerves selected from the group consisting of glioblastoma-derived neuronal growth factor, brain-derived neurotrophic factor, and neurotropin-3 in the human placenta's chorion or Wharton cord colloid-derived stem cells It may be differentiated by culturing in a medium containing a growth factor.
  • the method of differentiating stem cells derived from chorionic membrane or Wharton umbilical cord collagen into nerve cells and hair cells first separates the chorion or Wharton umbilical cord colloid from the placenta and separates the mesenchymal stem cells from the separated chorionic or wort umbilical cord collagen. Then, the isolated mesenchymal stem cells can be differentiated into neurons or hair cells, respectively, by treating nerve growth factors in the culture medium in which the cells grow.
  • the culture medium may be used as long as the medium for animal cell culture used in general, but is not limited thereto, DMEM, alpha-DMEM, Eagle's basal maxim, RPMI 1640 medium, NPBM (Neural progenitor) cell basal medium (SClonetics).
  • the neuronal growth factor used in the present invention for differentiating from chorionic or Wharton umbilical cord-derived mesenchymal stem cells into neurons or hair cells the glioblastoma-derived neuronal growth factor, brain-derived neurotrophic factor and neurotrophin-3
  • each nerve growth factor may be included at a concentration of 5 ng / ml to 15 ng / ml with respect to the total volume of the culture medium.
  • the medium also contains 1 to 3 weight percent (v / v) of B-27 additive, 1 to 3 mM L-glutamine, and antibiotic-antifungal agents (eg, penicillin-streptomycin), based on the total volume of the medium. Weight% (v / v) may be added further.
  • Fig. 2 it was confirmed that the cells are capable of self-proliferation by staining for BrdU, a marker of proliferating cells during cell division, and the myosin VII and TRPA, markers of hair cells, were also expressed (Fig. 5).
  • the neuronal markers NF and ⁇ III-tubulin and Glial cell markers MBP and S-100 were also expressed (Figs. 6 and 7).
  • 10ng / ml EGF and 20ng / ml bFGF were added to DMEM medium used as a conventional animal cell medium, followed by culturing, and then differentiated neural cell precursors were nested as neural markers. Immunofluorescence analysis showed that most of them were stained with Nestin and RT-PCT experiments using primers that could amplify the genes of the neural stem cell markers Nestin, BMP4 and BMP7. It was also confirmed that the genes of the marker were expressed in differentiated neuronal cell precursors (FIG. 9).
  • the present inventors were found that when cultured in chorion or Wharton's gelatin-derived mesenchymal stem cells in a medium to which EGF or bFGF is added, they differentiate into neuronal cell precursors, whereas GDNF, BDNF or When cultured in the medium to which NT-3 was added, it was found to differentiate into neurons and hair cells.
  • deafness is the most common disease in the world, and in the elderly after 65 years, about 30% suffer from such difficulties in everyday life.
  • peripheral sensory nerve cells, hair cells are lost due to the blockage of the afferent path, and the auditory nerve cells and cochlear ganglion cells are also lost over time.
  • the current method is to wear hearing aids to patients with hearing loss or to help patients through cochlear implant surgery.However, the development of neuronal cells of cochlear ganglion among cochlear implant patients In the case of deterioration or deterioration due to prolonged hearing loss, even if cochlear implantation is performed, there is a limit to the full recovery of hearing.
  • composition of the present invention can be used as a pharmaceutical composition for the prevention or treatment of hearing loss.
  • the pharmaceutically effective amount of neurons and hair cells differentiated from chorion-derived mesenchymal stem cells derived from chorionic membrane or Wharton cord colloid according to the present invention is 0.5 to 100 mg / day / kg body weight, preferably 0.5 to 5 mg / day / kg body weight.
  • the pharmaceutically effective amount may be appropriately changed depending on the degree of symptoms of hearing loss, the age, weight, health condition, sex, route of administration and duration of treatment of the patient.
  • Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid.
  • Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.
  • Other pharmaceutically acceptable carriers may be referred to those described in the following documents (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).
  • the pharmaceutical composition according to the present invention may be formulated in a suitable form according to methods known in the art together with the pharmaceutically acceptable carrier as described above.
  • the pharmaceutical composition of the present invention can be prepared in various parenteral or oral dosage forms according to known methods, and isotonic aqueous solution or suspension is preferable as an injectable formulation as a typical parenteral dosage form.
  • injectable formulations may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • each component may be formulated for injection by dissolving in saline or buffer.
  • formulations for oral administration include, but are not limited to, powders, granules, tablets, pills and capsules.
  • the present invention can provide a composition for the prevention and treatment of hearing loss, which comprises neuronal cell precursors, neurons and hair cells differentiated from chorionic or Wharton cord-derived stem cells.
  • the present invention provides a method for differentiating neuronal cell precursors, neurons and / or hair cells from chorionic or wort-cell-derived stem cells of human placental tissue and hearing loss comprising the differentiated neuronal cell precursors, nerve cells and / or hair cells. It provides a composition for the prevention or treatment of cancer, can be used in the field of cell replacement therapy and regenerative medicine for the treatment of hearing loss caused by damage of hair cells, and can be used as a material for the development of new drugs for the treatment of hearing loss. In addition, there is an effect that can be widely used in the field of basic research related to the treatment of hearing loss.
  • FIG. 1 shows stem cells isolated from chorionic membrane or wharf cord colloid according to one embodiment of the present invention, and shows the photograph of the shape of the cells under a microscope while subcultured.
  • A” and “C” show photographs of culture 5 of chorion-derived stem cells
  • B” and “D” show photographs of culture Day 5 of wharton medullary stem cells.
  • Figure 2 shows a microscopic picture of the shape of the cells according to the period of induction of differentiation of stem cells from the chorionic membrane or wharf cord colloid according to one embodiment of the present invention.
  • 3a to 3j show the results of confirming whether the cells separated from the chorionic membrane of the placenta are stem cells through FACS analysis using specific markers on the cell surface.
  • Figures 4a to 4j shows the results of confirming whether the cells isolated from the Wharton umbilical cord colloid of the placenta through FACS analysis using specific markers on the cell surface.
  • Figure 5 shows the results of performing the double-label immunofluorescence using the markers of myosin VIIA and TRPA1 to determine whether the differentiation into hair cells.
  • Figure 6 shows the results of performing the double-label immunofluorescence using the markers of NF and ⁇ III-tubulin to determine whether the differentiation into neurons.
  • Figure 7 shows the results of performing the double-label immunofluorescence using the markers of s100 and MBP to determine whether the differentiation into glial cells.
  • FIG. 8 shows a fluorescence image of the state before differentiation.
  • the upper row is before the differentiation of stem cells derived from Wharton umbilical cord, and the lower row is before the differentiation of stem cells derived from chorion.
  • Blue indicates counterstaining of nuclei with DAPI and green indicates proliferation markers stained with brdU.
  • Figure 9 shows the results of the differentiation and expression of neurons, hair cells and neuronal cell precursors from chorionic membrane or Wharton umbilical cord cells by RT-PCR method.
  • Undifferentiated cells is a pre-differentiation of stem cells derived from chorionic or wharton umbilical cord
  • Choorion MCSs is a result of stem cells derived from chorionic membranes differentiated according to the method of the present invention
  • “Wharton's jelly” Stem cells derived from the collagen are shown after differentiation according to the method of the present invention.
  • Example 2 Identification of adult stem cells derived from the isolated chorion or Wharton cord colloid
  • Example 2 In order to confirm that the cells derived from placental chorionic umbilical membrane or Wharton umbilical cord gel obtained in Example 1 were adult stem cells, a cell surface maker of stem cells was used. For this purpose, CD34, CD45, CD73, CD90, CD146, CD103, CD105 and HLADR (histocompatibility marker) antibodies were identified using flow cytometry (FACS Caliber, Becton Dickson, San Diego, CA) equipment.
  • flow cytometry FACS Caliber, Becton Dickson, San Diego, CA
  • the cells derived from the chorionic membrane or the wharf cord colloid are stem cells (FIGS. 2 and 3A to 3J).
  • the shape of the cells became longer as the number of subcultures increased. fibroblast like cell) was confirmed to change.
  • the mesenchymal stem cells derived from the chorion membrane or Wharton cord gelatin isolated and cultured in Example 1 were differentiated into neural cell precursors, hair cells and neurons by the following method.
  • the composition of the medium for differentiation from chorionic or Wharton cord-derived stem cells into neural cell precursors is shown in Table 1 below.
  • the composition was induced differently from the culture medium of the neuronal cell precursor culture medium.
  • the basal medium was used as a nerve cell culture medium (neurobasal medium), the neuronal growth factors of glioblastoma-derived nerve growth factor (Invitrogen), brain-derived neurotrophic factor (Invitrogen) and neurotropin-3 (Invitrogen) Neural system growth factors were used, and the composition of the medium used for the differentiation of hair cells and neurons is shown in Table 2 below.
  • Neuronal cell precursors, hair cells and neurons obtained by differentiating from chorional membrane or Wharton cord colloid-derived mesenchymal stem cells by the method of Example 3 were subjected to immunofluorescence staining, respectively. And neuronal cells.
  • BrdU is labeled on cells for 24 hours using BrdU (5-Bromo-2'-deoxy-uridine Labeling and Detection Kit, Roche, Indianapolis, IN), which is known as a marker for proliferating cells to identify specific proliferation. labeling).
  • BrdU 5-Bromo-2'-deoxy-uridine Labeling and Detection Kit
  • GFAP S-100
  • MBP glial markers ⁇ III-tubulin
  • NF NF
  • MAP2 nestin
  • neuronal markers myosin ⁇ A
  • TRPA1 Double staining was performed using.
  • the expression of ctbp2 was confirmed to simultaneously perform functional analysis of the hair cells.
  • DAPI was used for nuclear staining, and expression was counted using the Fluorescence Attached Microscope to count the cells expressed per cell count.
  • each of the stained cells were examined under a microscope.
  • neural cell precursors differentiated from chorion or Wharton's collagen-derived mesenchymal stem cells they gradually became spherical in shape as they induced differentiation into neural progenitor cells.
  • BrdU a marker of proliferating cells during cell division, was confirmed to be capable of self-proliferation, and markers of hair cells, myosin VII and TRPA, were also expressed (Fig. 5), NF and ⁇ III-tubulin neuronal markers and MBP and S-100, Glial cell markers, were also expressed (FIGS. 6 and 7).
  • the chorion membrane or Wharton cord colloid-derived stem cells according to the present invention are well differentiated into neural cell precursors, hair cells, and neurons.
  • Example 3 the expression of neural cell precursors, hair cells, and neurons was identified through RT-PCR of neural cell precursors, hair cells, and neurons differentiated from chorionic or Wharton cord-derived stem cells. That is, for this purpose, RNA was extracted from each of the cells after differentiation and subjected to polymerase chain reaction (PCR) through reverse transcription. Synthesized cDNA using BMP4 (Bone Morphology Protein 4), BMP7 primers of the genes used in the immunofluorescence staining method and denature the DNA at 94 °C for 5 minutes and reacted for 30 seconds at the binding temperature of each gene, 72 DNA synthesis and extension reaction were performed for 5 min.
  • BMP4 Ben Morphology Protein 4
  • GAPDH GlycerAldehyde-3-Phosphate DeHydrogenase
  • genes of nestin which are markers of stem cells, were expressed, and gene expression was also identified in inner ear development markers BMP4 and BMP7.
  • Myosin VIIA and TRPA1 which are markers of hair cells, and ⁇ III-, which are neuronal markers, were identified. It was confirmed that tubulin, MAP2, MBP, S-100 and GFAP genes are also expressed (Fig. 9).
  • the present inventors found that neural cell precursors, hair cells, and neurons were well differentiated from the chorional membrane or Wharton umbilical cord-derived mesenchymal stem cells.
  • the present invention relates to a method for differentiating neuronal cell precursors, neurons and / or hair cells from chorion or wharf cell-derived stem cells of human placental tissue, wherein the cell replacement therapy for treating hearing loss caused by hair cell damage And regenerative medicine.

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Abstract

La présente invention concerne un procédé pour différencier des cellules souches isolées du chorion placentaire humain ou de la gelée de Wharton, et plus précisément un procédé pour différencier des cellules souches dérivées du chorion ou de la gelée de Wharton en des précurseurs de cellules neurales, des cellules ciliées et des cellules nerveuses, le procédé comprenant la culture des cellules souches, isolées du chorion placentaire humain ou de la gelée de Wharton, dans un milieu de culture contenant un facteur de croissance des nerfs. La présente invention concerne en outre une composition pour prévenir ou traiter la perte d'audition, comprenant en tant que principes actifs les précurseurs de cellules neurales différenciées, des cellules ciliées et des cellules neurales.
PCT/KR2014/009485 2013-11-29 2014-10-08 Procédé pour différencier des cellules nerveuses et des cellules ciliées et des cellules souches mésenchymateuses dérivées du chorion placentaire ou de la gelée de wharton WO2015080376A1 (fr)

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