WO2015080376A1 - Method for differentiating nerve cells and hair cells from placental chorion or warthon's jelly-derived mesenchymal stem cells - Google Patents

Abstract

The present invention relates to a method for differentiating stem cells isolated from human placental chorion or Warthon's jelly and, more specifically, to a method for differentiating chorion or Warthon's jelly-derived stem cells into neural cell precursors, hair cells and nerve cells, the method comprising culturing stem cells, isolated from human placental chorion or Warthon's jelly, in a culture medium containing a nerve growth factor. In addition, the present invention relates to a composition for preventing or treating hearing loss, comprising the differentiated neural cell precursors, hair cells and neural cells as active ingredients.

Description

Method for Differentiating Neurons and Hair Cells from Placenta Chondrocytes or Wharton Umbilical Cord Collagen-derived Mesenchymal Stem Cells

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.

Conventionally, 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.

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.

In recent years, cell replacement therapy, 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.

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. Say. 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.

Meanwhile, mesenchymal stem cells (MSCs) are differentiated into bone, cartilage, adipose tissue, muscle, tendons, ligaments, and neural tissues, and thus are attracting attention as cells suitable for cell therapy. Currently, bone marrow is the most representative tissue from which mesenchymal stem cells can be obtained. However, 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. In addition, for bone marrow transplantation, there is a problem in that a donor does not show a graft-versus-host response because the antigenic phenotypes are matched through histocompatibility antigen comparison.

Therefore, recently, it is known that a large amount of stem cells exist in the cord blood (Umblical cord blood, UCB), and research on cell therapy using cord blood is being actively conducted. In particular, many attempts have been made to treat blood-related diseases through umbilical cord blood transplantation, and umbilical cord blood banks that freeze umbilical cord blood and preserve it in a usable state for several years for autograft treatment are being activated in Korea.

In addition, recently, studies for the isolation of mesenchymal stem cells from fetal tissues have been actively conducted. As a result, it has been found that there are abundant mesenchymal stem cells, but the use of fetal tissues for cell therapy Due to ethical limitations, there were limitations for use as cell therapy. Although 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.

On the other hand, 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. In addition, as the aging population has grown exponentially, the number of senile deafness has been increasing rapidly. 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.

In general, the inner ear in mammals is known to not regenerate sensory hair cells of already damaged or dead inner ear. Thus hearing impairment due to the death or weakening of sensory hair cells typically causes permanent hearing impairment. In addition, sensory neuronal deafness includes age-related loss (senile deafness), noise exposure, drug exposure (eg antibiotics and anti-cancer treatment), infections, genetic mutations (syndromes and non-syndromes), and autoimmune diseases. It may be caused by a number of factors including.

Currently, the use of external hearing aids and cochlear implants are being used as treatments for sensorineural hearing loss, both of which have somewhat limited therapeutic potential. In addition, recently, a method for regenerating sensory inner hair cells has been developed. International patent application PCT / US99 / 24829 discloses a method for stimulating regeneration of inner ear cells including sensory hair cells. However, the technique requires a very complex process and a long time, including the introduction of an endogenous cell nucleic acid molecule encoding a transcription factor to stimulate regeneration of the inner ear cell. Therefore, there is a need for a new technology that can effectively regenerate neurons and hair cells to treat hearing loss.

However, there have been no examples of differentiation and proliferation of neurons and hair cells from the placenta's chorion or Wharton's colloid-derived stem cells for the treatment of hearing loss.

Against this background, while the present inventors are studying a method for effectively treating hearing loss through regeneration of hair cells, neural cell precursors, nerve cells, and hair cells, which are important in the treatment of hearing loss, are placed in the placenta's chorional membrane or Wharton medullary glia mesenchyme. The present invention was completed by confirming that they can be differentiated from stem cells.

An object of the present invention comprises the step of culturing stem cells isolated from the chorionic membrane or wharf umbilical cord of human placenta in a medium containing a neural growth factor, the stem cells derived from the chorionic or wort umbilical cord collagen, neuronal cell precursors, hair cells and It provides a method of differentiating one selected from the group consisting of neurons.

Another object of the present invention for the prevention or treatment of hearing loss comprising one selected from the group consisting of neuronal cell precursors, hair cells and neurons differentiated from chorion-derived collagen-derived stem cells of human placenta It is to provide a composition.

Another object of the present invention is to administer to a subject with hearing loss a therapeutically effective amount of one selected from the group consisting of neuronal cell precursors, hair cells, and neurons differentiated from chorion-derived stem cells from human placenta chorion It is to provide a method of treating hearing loss.

As one embodiment for achieving the above object, the present invention comprises the step of culturing stem cells isolated from the chorionic membrane or wharf umbilical cord of the human placenta in a medium containing nerve growth factor, chorionic or wort umbilical cord-derived stem A method of differentiating a cell into one selected from the group consisting of neural cell precursors, hair cells and neurons.

Preferably, the neuronal growth factor (Epidermal Growth Factor, EGF), fibroblast growth factor (basic fibroblast growth factor, bFGF), glioblastoma-derived neurot growth factor (GDNF), brain Derived neurotrophic factor (Brain-derived neutrophin factor, BDNF) and neurotrophin-3 (Neurotrophin-3, NT-3) may be one or more selected from the group consisting of.

Also preferably, the medium further contains 1 to 3 wt% (v / v) of B-27 supplement, 1 to 3 mM L-glutamine and 0.5 to 2 wt% (v / v) of antibiotic. It may be.

Also preferably, the culturing may be to be cultured for 15 to 26 days.

Also preferably, the nerve growth factor may be an epithelial growth factor or a fibroblast growth factor, and may differentiate the chorionic or Wharton umbilical cord-derived stem cells into neural cell precursors.

More preferably, 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.

Also preferably, 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.

More preferably, the glioblastoma-derived nerve growth factor is 5-15 ng / ml in the medium, the brain-derived neurotrophic factor is 5-15 ng / ml in the medium, and the neurotropin-3 is 5-15 ng / in the medium. M may be included.

As another aspect, the present invention, 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.

In another aspect, the present invention provides a therapeutically effective amount for administering to a subject with deafness a therapeutically effective amount of one selected from the group consisting of neuronal cell precursors, hair cells, and neurons differentiated from chorion-derived stem cells from human placenta, It relates to a method of treating deafness comprising the step.

Preferably, the neuronal cell precursor may be differentiated by culturing the human placenta's chorion or Wharton's colloid-derived stem cells in a medium containing epithelial growth factor or fibroblast growth factor.

Also preferably, 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.

Hereinafter, the present invention will be described in more detail.

The present invention includes the step of culturing mesenchymal stem cells derived from the placenta's chorionic membrane or wharf cord colloid in a medium containing nerve growth factor, the neuron and It is to provide a method for differentiating into hair cells.

The placenta is made for the fetus during pregnancy and has a disc shape of 500g in weight, 15 ~ 20cm in diameter, and 2 ~ 3cm in thickness. One side of the placenta is in contact with the mother and the other is in contact with the fetus, and the space between the mother's blood contains the nourishment to the fetus. The placenta is composed of three layers of amnion, chorion, and decidual membrane. The chorionic membrane is a thin membrane between the amniotic membrane and the decidual membrane. Wharton umbilical cord is composed of cords (string-shaped structure connecting the fetus and placenta, umbilical cord). It is the main ingredient.

Meanwhile, in the present invention, stem cells were separated from the chorionic membrane or the wharf cord colloid (Example 1) among the constituents of the placenta, and differentiation of neurons and hair cells through culturing in a medium containing a specific growth factor. Conditions were established (Example 3).

In addition, the present inventors, using the placenta's chorionic membrane or Wharton's umbilical cord-derived mesenchymal stem cells for the purpose of treating a hearing impairment such as hearing loss, from the neuron and the inner ear sensor cells to the hair cells Differentiation of stem cells from the chorional membrane or Wharton umbilical cord colloid of placental tissue, and when the mesenchymal stem cells were cultured in a medium containing nerve growth factor, they differentiated into neurons and hair cells. Induction was confirmed (Examples 4 and 5).

Therefore, the present invention can provide a method of differentiating the chorion membrane or Wharton umbilical cord-derived stem cells of placental tissue into neurons and hair cells, and more specifically, the chorionic or Wharton umbilical cord-derived stem cells of human placental tissue It can be provided a method of differentiating chorion membrane or Wharton preparations-derived stem cells into neurons and hair cells, comprising culturing in a medium containing growth factors.

In addition, the method of differentiating stem cells derived from chorionic membrane or Wharton umbilical cord collagen into nerve cells and hair cells, according to the present invention, 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 method for obtaining stem cells from the chorionic membrane or Wharton umbilical cord may first separate the chorionic or wort umbilical cord from the placental tissue, and then obtain the stem cells from the chorionic or wort umbilical cord, which is known in the art. Any method may be used, for example, chorion or wharf colloids are isolated from human placental tissue samples, washed with Hanks' balanced salt solution to remove blood, and then chemically digested in 0.05% trypsin solution, followed by trypsin. After collecting the treated solution, it is cultured in a medium containing EGF to obtain a stem cell solution derived from chorionic membrane or Wharton umbilical cord isolated from the human placenta. In addition, the obtained stem cells can be repeated subculture until the stem cells are sufficiently amplified.

The chorion membrane or Wharton umbilical cord-derived stem cells obtained by the above method can be differentiated into neurons or hair cells, respectively, by treating and culturing the nerve growth factor 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 mediu, RPMI 1640 medium, NPBM (Neural progenitor) cell basal medium (SClonetics).

In addition, 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 One or more selected from the group may be used, and 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.

In addition, the culture may be carried out at a temperature of 36 to 38 ℃ and 4 to 6% carbon dioxide conditions for 15 to 26 days.

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.

Meanwhile, among the nerve growth factors, brain-derived neurotrophic factors are neurotrophin, which is most abundantly distributed in the brain, which promotes the growth of neurons, and the synthesis, metabolism, release of neurotransmitters and neurons. It is known to act to regulate the activity of.

In addition, the glioblastoma-derived nerve growth factor and neurotrophin-3 are known to act to promote the growth of glial cells, glial cells and neurons.

On the other hand, the inventors of the present invention as a result of examining the differentiation of neurons and hair cells by culturing stem cells isolated from the chorional membrane or wharf cord colloid of human placental tissue in a medium containing a specific growth factor according to the method of the present invention, It was confirmed that neural cell precursors, neural cells, and hair cells are well differentiated from the chorion membrane or the wharton umbilical cord-derived stem cells.

More specifically, according to one embodiment of the present invention, the differentiation of neurons and hair cells was induced by culturing the chorion or Wharton's colloid-derived stem cells in a medium containing nerve growth factors of GDNF, BDNF and NT-3. In order to confirm the differentiation of the neurons and hair cells, the cells were first stained with Brud5, which can identify specific proliferation, and then GFAP (Glial Fibrillary Acidic) as a glial marker for identifying each distinctly differentiated cell. Protein), S-100, and Myelin basic protein (MBP), beta III-tubulin, NF (Neurofilamen), and MAP2 (Microtubul-Associated Protein2) as neuronal markers; (nestin) was performed by fluorescence immunoassay using double staining using myosin ⅦA and TRPA1 (Transient receptor potential cation channel) as markers of hair cells.

Then, microscopic examination of each of the stained cells showed that neuronal cell precursors differentiated from chorion or Wharton's gelatin-derived mesenchymal stem cells were gradually spherical in shape as they induced differentiation into neural progenitor cells. (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 βIII-tubulin and Glial cell markers MBP and S-100 were also expressed (Figs. 6 and 7).

In addition, according to another embodiment of the present invention, after inducing differentiation of neurons and hair cells from the chorion or Wharton umbilical cord gel-derived stem cells in a medium containing nerve growth factors of GDNF, BDNF and NT-3, The degree of differentiation and expression of neurons and hair cells was examined by performing RT-PCR using primers capable of amplifying genes corresponding to markers that recognize cells.

As a result, the genes of nestin (nestin), which is a marker of stem cells, were expressed, and gene expression was also confirmed in the inner ear development markers BMP4 and BMP7, and markers of hair cells, Myosin ⅦA and TRPA1. And neuronal markers βIII-tubulin, MAP2, S-100 and GFAP genes were also expressed (Fig. 9).

Therefore, the present inventors, through the above results, when culturing choriocartilage-derived mesenchymal stem cells derived from chorional membrane or wharton umbilical cord cell-derived culture medium, that is, a medium containing a nerve growth factor, that is, GDNF, BDNF or NT-3, It was found that neurons and hair cells can be differentiated from mesenchymal stem cells.

In addition, the present inventors induced the differentiation of neuronal cell precursors from chorion or Wharton cord colloid-derived mesenchymal stem cells. Specifically, the differentiation into the neuronal cell precursors induces differentiation by adding EGF, which is an epithelial growth factor, and bFGF, which is a stromal fibroblast growth factor, to the medium for culturing the chorion or Wharton's gelatin-derived mesenchymal stem cells. It was.

Epidermal growth factor, which is a growth factor used to induce differentiation of neuronal cell precursors from the chorionic stem cell-derived mesenchymal stem cells, is derived from the submaxillary glands and the Brunner's gland, and the mesenchymal stem cells It is known to enhance differentiation of glial cells and the like. In addition, bFGF, a stromal fibroblast growth factor that can be obtained in most of the human body, is known to promote wound healing and cell differentiation.

According to one embodiment of the present invention, 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).

Therefore, through the above results, 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.

On the other hand, 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. When such deafness occurs, 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. In order to improve or treat hearing loss, 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.

Therefore, in order to overcome this limitation, the concept of nerve cell therapy for hearing recovery is emerging. Especially, if hair cells, which are peripheral sensory nerve cells that cause hearing loss, can be reproduced, the hearing loss can be treated more fundamentally.

The method of differentiating the chorion-derived mesenchymal stem cells derived from chorionic membrane or wort umbilical cord collagen into neurons and hair cells according to the present invention has an effect of differentiating and regenerating hair cells, which are auditory sensory cells that cause hearing loss, and thus can prevent or treat hearing loss. There is a characteristic.

Therefore, the present invention can provide a composition for the prevention or treatment of hearing loss, which comprises neurons and hair cells differentiated from chorion or Wharton medullary cells derived from mesenchymal cells as an active ingredient.

In addition, the composition of the present invention can be used as a pharmaceutical composition for the prevention or treatment of hearing loss.

In the present invention, the "hearing loss" refers to a state in which hearing is degraded or lost due to impairment of hearing organs, and may include both a syndrome and a nonsyndromic form. The syndrome can be further subdivided into three types, namely 1) prenegative hearing loss due to middle and outer ear defects with or without sensory nerve components associated with hearing impairment, and 2) sensory nerve components associated with hearing impairment. Middle-to-near hearing loss with or without middle ear defects, and 3) sensorineural hearing loss due to cochlear or retrocochlear defects. In addition, the non-syndrome refers to hereditary deafness and can be classified into DFN, DFNA and DFNB according to their genetic pattern, which means X chromosome-associated, autosomal dominant and autosomal recessive delivery modalities, respectively.

A composition for the prevention or treatment of hearing loss according to the present invention may include a neuronal and hair cells differentiated from pharmaceutically effective amounts of chorion or Wharton's colloid-derived mesenchymal stem cells alone or one or more pharmaceutically acceptable carriers and excipients. Or diluents. The pharmaceutically effective amount in the above means an amount sufficient to prevent, ameliorate and treat hearing loss.

In addition, the present invention includes the step of administering to a subject with deafness a therapeutically effective amount of one selected from the group consisting of neuronal cell precursors, hair cells, and neurons differentiated from chorion-derived stem cells from the human placenta's chorion membrane Provides a treatment for hearing loss. As used herein, the term “therapeutically effective amount” or “effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to the treatment of hearing loss, and the effective dose level refers to the individual type, condition and severity, age By the person skilled in the art, depending on factors well known in the art, gender, type of infected bacteria, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of administration, duration of treatment, concurrently used drugs and other medical fields May be appropriately selected.

The neuronal cell precursor may be differentiated by culturing the human placenta's chorion membrane or Wharton cord colloid-derived stem cells in a medium containing epithelial growth factor or fibroblast growth factor.

The hair cell or the neuron may comprise one or more nerve growth factors 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 the medium.

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. . However, 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.

The pharmaceutical composition according to the present invention may further include a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and that, when administered to a human, typically does not cause allergic or similar reactions, such as gastrointestinal disorders, dizziness, and the like. Pharmaceutically acceptable carriers include, for example, carriers for oral administration such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like, and parenteral administration such as water, suitable oils, saline, aqueous glucose and glycols, and the like. And the like may further comprise stabilizers and preservatives. 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. That is, 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. For example, each component may be formulated for injection by dissolving in saline or buffer. In addition, formulations for oral administration include, but are not limited to, powders, granules, tablets, pills and capsules.

Pharmaceutical compositions formulated in such a manner can be administered in a effective amount via several routes, including oral, transdermal, subcutaneous, intravenous or intramuscular. As used herein, the term 'effective amount' refers to an amount exhibiting a prophylactic or therapeutic effect when administered to a patient.

The dosage of the pharmaceutical composition according to the present invention may be appropriately selected depending on the route of administration, subject to administration, age, gender weight, individual difference and disease state. Preferably, the pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the degree of disease, preferably 1 to 10,000 g / weight kg / day, more preferably 10 to 1000 mg / weight kg The effective dose of / day may be repeated several times a day. In addition, the composition for the prevention or treatment of hearing loss according to the present invention may be administered in parallel with a known compound having the effect of preventing, improving or treating hearing loss.

Therefore, 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, and "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 βIII-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.

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, "Chorion 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.

10 shows the results of functional analysis of differentiation induced hair cells.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, the present invention is not limited by the following examples.

Example 1 Isolation and Culture of Human Placental Chorionic Chorionic Verticular Glia Stem Cells

The following experiments performed in the present invention were performed in compliance with IRB approval (Clinical Study Management Regulation KC08CIMI0344), and the chorionic or wort umbilical cord from the placenta, which was first collected under the consent of the mother for separation of the chorionic or wort umbilical cord from the placenta. The colloid and placenta were separated and washed with Hanks' balanced salt soultion to remove blood. Then, the chorionic membrane or Wharton umbilical cord was washed twice with PBS and then chopped to separate stem cells from the chorionic or Wharton cord colloid.

After immersion in 0.25% trypsin EDTA and incubated for 1 hour in a water bath at 37 ℃ 100rpm. After 10ml of culture medium containing FBS, an inhibitor of EDTA, pipetting about 20 times and centrifugation at 1000rpm for 5 minutes, and then separated from the culture medium containing 10ng / ml EGF for culturing stem cells. Cells were cultured. After 5 days of culture, the cells in the culture medium were subjected to cell separation again using trypsin to induce subculture and differentiation, thereby obtaining stem cells derived from chorionic or wort umbilical cord colloid.

Example 2. Identification of adult stem cells derived from the isolated chorion or Wharton cord colloid

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.

As a result, it was confirmed that the cells derived from the chorionic membrane or the wharf cord colloid are stem cells (FIGS. 2 and 3A to 3J). In addition, as a result of microscopic observation of the cell shape according to the increase in the number of subcultures during subculture of mononuclear cells isolated from the chorionic or wort umbilical cord, the shape of the cells became longer as the number of subcultures increased. fibroblast like cell) was confirmed to change.

Example 3 Differentiation of Neural System Precursors, Hair Cells and Neurons from Stem Cells Derived from Human Chorionic Membrane or Wharton Cord Gelatin

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.

3-1. Differentiation into Nervous System Cell Precursors

Differentiation of the chorion membrane or Wharton umbilical cord-derived stem cells into neural cell precursors induced differentiation into neural cell precursors by treating neural growth factors in the culture medium of the stem cells, wherein the culture medium for differentiation into neural cell precursors Was treated with 20ng / ml of EGF (Invitrogen) and 10ng / ml of basic fibroblast growth factor (Invitrogen), and cultured for 21 days in a medium containing the factors, and 10ng / ml of bFGF at 3 days intervals. Was added to the culture solution three times and cultured. 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.

Table 1

Culture medium for differentiating neural cell precursors
badge	DMEM: F12 (1: 1)
B-27 supplement	1 ml
L-Glutamine	2mM
EGF	10ng / ml
bFGF	20ng / ml
1% antibiotic-antimycotic
Total volume	50 ml

3-2. Differentiation into hair cells and neurons

In order to differentiate the chorion-derived mesenchymal stem cells from the chorional membrane or wort umbilical cord cells into differentiation of the hair cells and the nerve cells, 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.

TABLE 2

Culture medium for hair cell and neuron differentiation
badge	Neurobasal medium
B-27 supplement	1 ml
L-Glutamine	2 mM
BDNF
	10 ng / ml
GDNF
	10 ng / ml
NT3
	10 ng / ml
1% Penicilin-Streptomycin
Total volume	50 ml

Example 4 Expression Analysis of Neural System Precursors, Hair Cells and Neurons by Immunofluorescence Staining

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.

First, 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). Afterwards, GFAP, S-100, MBP, glial markers βIII-tubulin, NF, MAP2, nestin, neuronal markers, myosin 유 A, TRPA1 Double staining was performed using. In addition, 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.

As a result, each of the stained cells were examined under a microscope. In the case of 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. It was confirmed (Fig. 2), 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 βIII-tubulin neuronal markers and MBP and S-100, Glial cell markers, were also expressed (FIGS. 6 and 7).

Therefore, it can be seen from the above results that 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 5 Expression of Nervous System Precursors, Hair Cells, and Neurons Using RT-PCR

In 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 ℃ 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. GAPDH (GlycerAldehyde-3-Phosphate DeHydrogenase) was used as the house keeping gene, and then expression was confirmed using 2% agarose gel. The expressed genes were identified by bands using an Image Analysis System (Bio-Rad, Herculesus, CA).

As a result, 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 βIII-, which are neuronal markers, were identified. It was confirmed that tubulin, MAP2, MBP, S-100 and GFAP genes are also expressed (Fig. 9).

Therefore, 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.

Example 6 Functional Analysis of Differentiation-Induced Hair Cells (FIG. 10)

The method of Example 3 was performed by immunofluorescence staining on hair cells obtained by differentiating from chorion membrane or Wharton umbilical cord-derived mesenchymal stem cells to determine whether the differentiated cells are hair cells. In order to determine the differentiation into hair cells, the antibodies were identified using the antibodies suitable for the characteristics of the synaptic sites. Specifically, their expression was confirmed using antibodies to presynaptic markers (CtBP2, VGLUT3) and postsynaptic markers (GluR subtybe). DAPI was used for nuclear staining, and expression was counted using a Fluorescence Attached Microscope to count cells expressed per cell count.

As a result, it was confirmed that presynaptic markers (CtBP2 and VGLUT3) and postsynaptic markers (GluR subtybe), which are synaptic markers of hair cells, are expressed (FIG. 10).

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.

Claims (14)

1. Cultivating stem cells from the chorion or warton's jelly of the human placenta in culture with a medium containing nerve growth factors, the chorion or wort umbilical cord-derived stem cells are neuronal cell precursors, hair Differentiating to one selected from the group consisting of cells and neurons.
2. The method according to claim 1,

   The neuronal growth factor (Epidermal Growth Factor, EGF), fibroblast growth factor (basic fibroblast growth factor, bFGF), glioblastoma-derived neurotprophin factor (GDNF), brain-derived neurotrophic factor (Brain-derived neutrophin factor, BDNF) and neurotrophin-3 (Neurotrophin-3, NT-3) is at least one selected from the group consisting of.
3. The method according to claim 1,

   The medium further comprises 1-3 wt% (v / v) of B-27 supplement, 1-3 mg of L-glutamine and 0.5-2 wt% (v / v) of antibiotic. .
4. The method according to claim 1,

   Wherein said culturing is incubated for 15 to 26 days.
5. The method according to claim 1,

   The nerve growth factor is epithelial growth factor or fibroblast growth factor, and the method of differentiating stem cells derived from chorionic membrane or wort umbilical cord collagen into neuronal cell precursors.
6. The method according to claim 5,

   The epithelial growth factor is 5 to 15 ng / ㎖ in the medium, the fibroblast growth factor is 15 to 25 ng / ㎖ in the medium.
7. The method according to claim 1,

   The nerve growth factor is at least one selected from the group consisting of glioblastoma-derived nerve growth factor, brain-derived neurotrophic factor, and neurotropin-3, and differentiates the chorionic or Wharton umbilical cord-derived stem cells into hair cells or neurons. Way.
8. The method according to claim 7,

   The glioblastoma-derived neuronal growth factor is 5 to 15 ng / ml in the medium, the brain-derived neurotrophic factor is 5 to 15 ng / ml in the medium, and the neurotrophin-3 is 5 to 15 ng / ml in the medium. Way to be.
9. A composition for preventing or treating hearing loss, comprising one or more selected from the group consisting of neuronal cell precursors, hair cells, and neurons differentiated from the chorionic membrane or Wharton umbilical cord-derived stem cells of the human placenta.
10. The method according to claim 9,

    Wherein the neuronal cell precursor is differentiated by culturing the human placenta's chorionic membrane or wharf cord collagen-derived stem cells in a culture medium containing epithelial growth factor or fibroblast growth factor, the composition for the prevention or treatment of hearing loss.
11. The method according to claim 9,

    The hair cell or the neuron may comprise one or more nerve growth factors 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. A composition for preventing or treating hearing loss, which is differentiated by culturing in a medium.
12. A method for the treatment of hearing loss, comprising administering to a subject with hearing loss a therapeutically effective amount of one selected from the group consisting of neuronal cell precursors, hair cells, and neurons differentiated from chorion-derived stem cells from the human placenta.
13. The method according to claim 12,

    Wherein the neuronal cell precursor is differentiated by culturing the human placenta's chorionic membrane or Wharton umbilical cord-derived stem cells in a culture medium containing epithelial growth factor or fibroblast growth factor, deafness.
14. The method according to claim 12,

    The hair cell or the neuron may comprise one or more nerve growth factors 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. A method for treating hearing loss, which is differentiated by culturing in a medium.

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