KR20150062817A - Method of inducing differentiation of mesenchymal stem cell derived from chorion or warthon's jelly isolated from human term placenta into neuron and hair cell - Google Patents

Abstract

The present invention relates to a method for differentiating stem cells isolated from the chorion or warthon′s jelly of the human placenta and, more specifically, to a method for differentiating stem cells derived from the chorion or warthon′s jelly into nervous cell precursors, hair cells, and nerve cells. The method comprises a step of culturing stem cells isolated from the chorion or warthon′s jelly of the human placenta in a culture medium including nerve growth factors. Moreover, the present invention relates to a composition for preventing or treating bradyecoia, wherein the differentiated nervous cell precursors, hair cells, and nerve cells are included as active ingredients.

Description

[0001] The present invention relates to a method for differentiating a mesenchymal stem cell derived from a mesenchymal stem cell of a placenta from a mesenchymal stem cell of a placenta,

The present invention relates to a method of differentiating stem cells isolated from a chorion or warthon's jelly of a human placenta, and more particularly, to a method of differentiating stem cells isolated from a chorion or warthon's jelly of a human placenta, The present invention relates to a method for differentiating chorionic villi or whiteness cord-derived stem cells into neural cell precursors, hair cells, and neurons, comprising culturing the cells in a medium containing a nerve growth factor. The present invention also relates to a composition for preventing or treating hearing loss comprising the differentiated neural precursor cell, hair cell, and nerve cell as an active ingredient.

Conventionally, organ transplantation and gene therapy have been proposed for the treatment of human intractable diseases, but there has been little practical application due to lack of immunity, lack of supply or lack of knowledge about vector development or disease genes.

Thus, interest in stem cell research has been heightened, and it has been recognized that all stem cells capable of forming all organs through proliferation and differentiation can solve root diseases as well as treatment of most diseases. In addition, many scientists have suggested the possibility of applying stem cells in various ways, ranging from almost all the long-term regeneration of the human body to treatment of intractable Parkinson's disease, various cancers, diabetes and spinal cord injury.

In addition, in recent years, when cells are destroyed or damaged by diseases, cell replacement therapy that supplies cells from the outside has been suggested as an effective treatment method. In particular, stem cells capable of proliferation and differentiation, (stem cells) are in the limelight.

Stem cells with various functions are cells that are differentiated into cells that constitute the tissue. They are capable of infinite proliferation in undifferentiated state, and are capable of differentiating 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 astrocyte, oligodendrocyte, or Schwann cell. Neural stem cells are differentiated into neural cells, such as neurons or glias, through the stages of neural progenitor cells or glioma precursor cells that produce specific neural cells.

Meanwhile, mesenchymal stem cells (MSCs) can be differentiated into bone, cartilage, adipose tissue, muscles, tendons, ligaments, nerve tissue, etc., and are attracting attention as cells suitable for cell therapy. At present, bone marrow is one of the most representative origins of mesenchymal stem cells. However, the mesenchymal stem cells present in the bone marrow are limited in their application range due to their limited differentiation and proliferative capacity, and because of the limitation derived from the bone marrow, several steps are required and the procedure is complicated, , Accompanied by mental and physical pain. In addition, for bone marrow transplantation, there is a problem that a donor that does not show a graft-versus-host response due to the coincidence of antigenic phenotype through comparison of histologically compatible antigens is a problem.

Therefore, recently, it has been known that a large amount of stem cells are present in umbilical cord blood (UCB), and cell therapy using cord blood is being actively studied. In particular, attempts have been made to treat blood related diseases clinically through cord blood transplantation, and cord blood banks, which freeze cord blood for autotransplantation therapy and preserve it in a usable state after a few years, have been activated in Korea.

Recently, studies on 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 tissue for cell therapy Because of the ethical limitations, there was a limit to use as a cell therapy agent. Although mesenchymal stem cells were isolated from umbilical cord blood as a source of fetal MSCs, the number of the mesenchymal stem cells was very small and the proliferation was not good. Therefore, there is a need for a new method for separating and culturing stem cells, which is easy to proliferate and can be cultured as a cell therapy agent.

On the other hand, if the hearing loss, which is one of the human senses, is lost, personally, it causes obstacles in daily life and causes a great loss in socioeconomic environment. Hearing loss is not caused only by hearing impairment, but when serious hearing loss occurs before language acquisition, normal language development is disturbed and speech disorder accompanies problems. In addition, recently, the number of elderly people with hearing loss has increased rapidly as the number of elderly people increases exponentially. Cruel hearing loss is one of the three major geriatric diseases with hypertension and degenerative arthritis (Cruickshanks et al, 1998) (Gates et al., 1990). Most of the senile hearing loss is sensory and neural, which is caused by degenerative changes of auditory hair cells and spiral ganglia .

In general, it is known that the dermis in mammals does not regenerate sensory hair cells of damaged or dead inner ear. Thus, hearing loss due to the death or weakening of sensory hair cells typically results in permanent hearing impairment. In addition, sensory neural deafness can also be associated with aging-related loss (senile hearing loss), noise exposure, drug exposure (e.g., antibiotic and anti-cancer therapy), infection, genetic mutations (syndromic and non- And may be caused by a number of factors including immune disorders.

Currently, the use of external hearing aids and doorframes are used as treatments for sensory neuronal hearing loss, both of which have only a limited therapeutic potential. In addition, recently, a method for regenerating sensory innate hair cells has been developed. International Patent Application PCT / US99 / 24829 discloses a method for stimulating regeneration of inner ear cells containing sensory hair cells. However, this technique requires a very complicated process and a long time, including the step of introducing the inner cell nucleic acid molecule encoding the transcription factor so as to stimulate regeneration of the inner cell. Therefore, there is a demand for a new technique for effectively regenerating nerve cells and hair cells to treat hearing loss.

However, until now, there have been no examples in which neural cells and hair cells are differentiated and proliferated from the chorion of the placenta or stem cells derived from Warton umbilical cord as a cell therapy agent for the treatment of hearing loss.

Under these circumstances, the present inventors have studied how to effectively treat deafness through the regeneration of hair cells. In the treatment of deafness, the neural precursor, nerve cell, and hair cell which are important in the treatment of hearing loss are transferred to the placental chorionic membrane or the umbilical cord- The present inventors have completed the present invention by confirming that they can differentiate from stem cells.

It is an object of the present invention to provide a method for culturing a chorionic membrane or a whitened cord-derived stem cell derived from a chorionic membrane of a human placenta or a stem cell from a whitish umbilical cord colony in a medium containing a nerve growth factor, Neuronal cells, and the like.

It is another object of the present invention to provide a method for preventing or treating hearing loss, which comprises, as an active ingredient, one selected from the group consisting of a neural precursor cell, a hair cell and a neuron cell differentiated from a chorionic membrane of a human placenta or stem cell derived from a whitish umbilical cord To provide a composition.

In accordance with one aspect of the present invention, there is provided a method for culturing a chorionic membrane or a whitening cord-derived stem, comprising culturing a stem cell isolated from a chorionic membrane of a human placenta or a whitish umbilical cord colloid in a medium containing a nerve growth factor The present invention relates to a method of differentiating a cell into one selected from the group consisting of a neural cell precursor, a hair cell, and a neuron cell.

Preferably, the nerve growth factor is selected from the group consisting of Epidermal Growth Factor (EGF), basic fibroblast growth factor (bFGF), glioblast-derived neurotrophin factor (GDNF) A neurotrophin-3, and a neurotrophin-3 (NT-3) may be selected from the group consisting of Brain-derived neurotrophin (BDNF) and Neurotrophin-3.

Also preferably, the medium further comprises 1-3% (v / v) B-27 supplement (B-27 supplement), 1-3 mM L-glutamine and 0.5-2% (v / v) antibiotic .

Also preferably, the step of culturing may be performed for 15 to 26 days.

Also preferably, the nerve growth factor is an epithelial cell growth factor or fibroblast growth factor, and may be to differentiate chorionic villus or whitish cord-derived stem cells into neural cell precursors.

More preferably, the epithelial growth factor is contained in the medium in an amount of 5 to 15 ng / ml, and the fibroblast growth factor is contained in the medium in an amount of 15 to 25 ng / ml.

Preferably, the nerve growth factor is at least one selected from the group consisting of neurotrophic factor derived from myoblast-derived neurotrophic factor, neurotrophin and neurotrophin-3, and the stem cell derived from chorionic villus or whitish cord is referred to as a hair cell or a neuron It may be to differentiate.

More preferably, the neurotrophin-3 is added to the medium in an amount of 5 to 15 ng / ml, the neurotrophic factor in the medium is 5 to 15 ng / ml in the medium, Ml. ≪ / RTI >

In another aspect, the present invention provides a method for preventing or treating deafness, comprising an effective ingredient selected from the group consisting of a neural precursor cell, a hair cell, and a neuron cell differentiated from a chorionic membrane of a human placenta or a stem cell derived from a whitish cord colony ≪ / RTI >

Preferably, the neural progenitor cell precursor may be differentiated by culturing human placental chorionic villi or whitish cord colony-derived stem cells in a medium containing an epithelial cell growth factor or fibroblast growth factor.

Preferably, the hair cell or the neuron cell further comprises at least one nerve growth factor selected from the group consisting of neuroblastoma derived neurotrophic factor, brain-derived neurotrophin and neurotrophin-3, It may be differentiated by culturing in a medium containing the factor.

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

The present invention relates to a method for culturing mesenchymal stem cells derived from a placenta chorionic membrane or a whitish umbilical cord colloid in a medium containing a nerve growth factor, And to provide a method of differentiating into a hair cell.

The placenta is made for the fetus during pregnancy. It is 500g in weight, 15-20cm in diameter, and 2 ~ 3cm thick. One side of the placenta touches the mother and the other side of the placenta touches the fetus. The placenta consists of three layers of amniotic membrane, chorionic membrane and decidual membrane. The chorionic membrane is a thin membrane between the amniotic membrane and the decidual membrane. The whiteness colloid constitutes the umbilical cord (a cord-like structure connecting the placenta and the placenta) It is the main ingredient.

In the present invention, stem cells are separated from the placenta constituting the chorionic membrane or the whitish umbilical colloid (Example 1), and then cultured in a culture medium containing a specific growth factor to differentiate neurons and hair cells (Example 3).

In addition, the present inventors have developed a method for treating a hearing-impaired disease such as hearing loss by using a placental chorionic membrane or a whitish umbilical cord-derived mesenchymal stem cell, which has not been tried so far, And differentiation conditions were established to separate stem cells from chorionic villi or whitish umbilical cord colloid of placenta tissue. When the mesenchymal stem cells were cultured in a medium containing nerve growth factor, they were differentiated into nerve cells and hair cells (Examples 4 and 5).

Therefore, the present invention can provide a method for differentiating chorionic villi of a placental tissue or a stem cell of a whitish cord colloid into a neuron and a hair cell, and more particularly, to a method for differentiating a chorion of a human placental tissue, A method for differentiating chorionic villi or whitened umbilical cord-derived stem cells into neuronal cells and hairy cells comprising culturing in a medium containing growth factors can be provided.

The method for differentiating chorionic villus or whitening cord-derived stem cells according to the present invention into neurons and hair cells comprises first separating chorionic villus or whitish umbilical cord from the placenta and extracting mesenchymal stem cells from the separated chorion or whitish umbilical cord colony And the isolated mesenchymal stem cells can be differentiated into neurons or hair cells by treating nerve growth factor in the culture medium in which the cells grow.

The method of obtaining stem cells from the chorionic membrane or the whitetecontaining colloid may first be to isolate the chorion or whitish umbilical colloid from the placental tissue and then obtain the stem cells from the chorion or whitish umbilical colloid, For example, the chorioamnion or whitening umbilical colloid is separated from a human placenta tissue sample, washed with Hanks' balanced salt solution to remove blood, subjected to chemical decomposition in 0.05% trypsin solution, After collecting the trypsin-treated solution, it is cultured in a medium containing EGF to obtain a chorionic membrane or a stem cell solution derived from a whitish umbilical cord isolated from a human placenta. In addition, the obtained stem cells can be repeatedly subcultured until the stem cells are sufficiently amplified.

The chorionic villi or the stem cell derived from the whitetecritic cord obtained by the above method can be differentiated into neurons or hair cells by treating and culturing the nerve growth factor in the culture medium in which the cells grow.

The culture medium may be any conventional culture medium for animal cell culture, including, but not limited to, DMEM, alpha-DMEM, Eagle's basal medium, RPMI 1640 medium, NPBM (Neural progenitor cell basal medium: SClonetics).

In addition, the nerve growth factors used in the present invention for differentiating into mesenchymal stem cells from chorionic villi or whitetum cord derived mesenchymal stem cells include neurotrophic factors derived from myoblast-derived nerve growth factor, brain derived neurotrophin and neurotrophin-3 One or more selected from the group can be used, and each nerve growth factor may be included at a concentration of 5 ng / ml to 15 ng / ml based on the total volume of the culture medium.

Further, the culturing can be carried out at a temperature of 36 to 38 ° C and 4 to 6% carbon dioxide condition for 15 days to 26 days.

In addition, the culture medium may contain 1 to 3% by weight (v / v) of a B-27 additive, 1 to 3 mM of L-glutamine, and 0.5 to 2% of an antibiotic-antifungal agent (e.g. penicillin-streptomycin) (V / v). ≪ / RTI >

Among the above-mentioned nerve growth factors, brain-derived neurotrophic factor is neurotrophin, which is most abundantly distributed in the brain, and acts to promote the growth of neurons. Neurotrophin synthesis, metabolism, And the like.

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

Meanwhile, the present inventors investigated whether the stem cells isolated from the chorionic villi of the human placenta tissue or the whitish umbilical cord colony differentiated into neurons and hair cells by culturing in a medium containing specific growth factors according to the above-described method of the present invention. As a result, It was confirmed that neural precursor cells, nerve cells and hair cells were well differentiated from stem cells of chorionic villus or whitum umbilical cord.

More specifically, according to one embodiment of the present invention, differentiation of neurons and hair cells was induced by culturing chorionic villus or whitish cord-derived stem cells in a medium containing nerve growth factor of GDNF, BDNF and NT-3 To confirm the differentiation of the neurons and hair cells, the cells were firstly stained with Brud5, which is capable of confirming specific proliferation. Then, for identification of differentiated cells, GFAP (Glial Fibrillary Acidic Protein), S-100 and MBP (Myelin basic protein), beta III-tubulin, NF (Neurofilamen) and MAP2 (Microtubul-Associated Protein 2) as neuronal markers, (myosin) VIIA and TRPA1 (transient receptor potential cation channel) as markers of hairy cells were used for the fluorescence immunoassay using double staining.

Then, each of the stained cells was examined by a microscope. As a result, in the case of the neural progenitor cells differentiated from the chorionic villi or the mesenchymal stem cells derived from the whitish cord, they gradually become spherical in shape as they induce differentiation into neural progenitor cells (Fig. 2). It was confirmed that BrdU, which is a marker of cell proliferating during cell division, is a cell capable of proliferation by itself, and myosin VII and TRPA, which are markers of hair cell, are also expressed 5), neural cell markers NF and βIII-tubulin, and glial cell markers MBP and S-100 were also expressed (FIGS. 6 and 7).

According to another embodiment of the present invention, the differentiation of neurons and hair cells from chorionic villi or whitened cord-derived stem cells is induced in a medium containing nerve growth factor of GDNF, BDNF and NT-3, RT-PCR was performed using a primer capable of amplifying a gene corresponding to a marker recognizing a cell, and the degree of differentiation and expression of neuron and hair cell were examined.

As a result, the gene of nestin, which is a marker of stem cells, was expressed, and the expression of genes of inner ear development markers BMP4 and BMP7 was also confirmed, and markers of hair cell myosin VIIA and TRPA1 And neural cell markers such as? III-tubulin, MAP2, S-100 and GFAP genes were also expressed (FIG. 9).

Therefore, the present inventors have found that when the chorionic villus or the umbilical cord-derived mesenchymal stem cells are cultured in a medium containing nerve growth factor, that is, a medium containing GDNF, BDNF or NT-3, It was found that the mesenchymal stem cells can differentiate into neurons and hair cells.

Furthermore, the present inventors induced the differentiation of neural cell precursors from chorionic villus or umbilical cord-derived mesenchymal stem cells. Specifically, the differentiation into the neural precursor cells can be carried out by introducing EGF, which is an epithelial cell growth factor, and bFGF, a fibroblast growth factor, into the culture medium for culturing the chorionic villus or whitening cord-derived mesenchymal stem cells, Respectively.

The epithelial growth factor, which is a growth factor used to induce the differentiation of neural cell precursors from the chorionic villi or whiteness cord-derived mesenchymal stem cells, is derived from submaxillary glands and brunner's glands, And it is known to promote differentiation of glial cells and the like. In addition, bFGF, which is a 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, 10 ng / ml of EGF and 20 ng / ml bFGF are added to DMEM medium used as an ordinary animal cell culture medium, and then the differentiated neural cell precursor is added to nerve system marker nestin Immunofluorescence analysis revealed that nestin was mostly expressed and stained. RT-PCT experiments using primers capable of amplifying the genes of nestin, BMP4 and BMP7, which are neural stem cell markers, It was confirmed that the genes of the markers were all expressed in differentiated neural precursor cells (FIG. 9).

Therefore, the present inventors have found that mesenchymal stem cells derived from chorionic villus or whitish cord are differentiated into neural cell precursors when cultured in a medium supplemented with EGF or bFGF, whereas GDNF, BDNF or When cultured in medium supplemented with NT-3, it was differentiated into neurons and hair cells.

On the other hand, hearing loss is the most common disease worldwide, and about 30% of elderly people after age 65 suffer from many difficulties in their everyday lives. When such hearing loss occurs, the peripheral nervous cells, which are peripheral sensory nerve cells due to the blockage of the central nervous system, are lost, and the auditory nerve cells and the cells of the womb ganglia are also lost over time. The current methods used to improve and treat hearing loss include helping patients with hearing loss by wearing a hearing aid or cochlear implants, but the development of neuronal cells in the ganglion ganglia And the degeneration of long-term hearing loss, the cochlear implantation of the cochlear implants is limited.

To overcome these limitations, the concept of nerve cell therapy for hearing recovery is emerging. In particular, if we can regenerate the peripheral nerve cell, which is the cause of deafness, we can treat the deafness more fundamentally.

The method for differentiating mesenchymal stem cells from chorionic villus or whitetum cord-derived mesenchymal stem cells into neurons and hair cells according to the present invention has the effect of differentiating and regenerating hair cells, which are auditory sensory cells responsible for deafness, Feature.

Therefore, the present invention can provide a composition for preventing or treating hearing loss comprising neuron and hair cell differentiated from chorionic villus or whitish cord-derived mesenchymal stem cells as an effective ingredient.

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

In the present invention, the term "hearing loss" refers to a state of hearing loss or loss due to a disorder of the auditory organ, and may include both syndromic and nonsyndromic forms. The syndrome pattern can be further subdivided into three categories: 1) full-tone hearing loss due to middle and outer ear defects with or without sensory neurons associated with hearing impairment, 2) sensory neurons associated with hearing impairment And 3) sensory nerve impairment due to cochlear or retrocochlear defects. In addition, the non-syndrome refers to hereditary deafness and can be classified into DFN, DFNA, and DFNB depending on their genetic form, and these mean X chromosome-associated, autosomal dominant and autosomal recessive transmission patterns, respectively.

The composition for preventing or treating hearing loss according to the present invention may comprise a naturally occurring cell or a hair cell differentiated from a pharmacologically effective chorionic membrane or a mesenchymal stem cell derived from a whitish cord colony alone or in combination with one or more pharmaceutically acceptable carriers, Or a diluent. A pharmaceutically effective amount as used herein refers to an amount sufficient to prevent, ameliorate, and treat hearing loss.

The pharmacologically effective amount of neuron and hair cell differentiated from the chorioamnion or whitening cord-derived mesenchymal stem cells according to the present invention is 0.5 to 100 mg / day / body weight kg, preferably 0.5 to 5 mg / day / body weight kg . However, the pharmaceutically effective amount may be appropriately changed depending on the symptom of the hearing loss, the patient's age, body weight, health condition, sex, administration route and treatment period.

The pharmaceutical composition according to the present invention may further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" as used herein refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to humans. Pharmaceutically acceptable carriers include, for example, carriers for oral administration such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid and the like, water for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycols And may further contain 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 can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995). The pharmaceutical composition according to the present invention, together with a pharmaceutically acceptable carrier as described above, may be formulated into a suitable form according to a method known in the art. That is, the pharmaceutical composition of the present invention can be prepared in various parenteral or oral administration forms according to known methods, and isotonic aqueous solutions or suspensions are preferred for injection formulations typical of parenteral administration formulations. The 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.

The pharmaceutical composition formulated as described above may be administered in an effective amount through various routes including oral, transdermal, subcutaneous, intravenous, or muscular. The term " effective amount " as used herein refers to an amount that shows a preventive or therapeutic effect when administered to a patient. The dosage of the pharmaceutical composition according to the present invention can be appropriately selected depending on the route of administration, subject to be administered, age, gender, individual difference, and disease state. Preferably, the pharmaceutical composition of the present invention may contain the active ingredient in an amount of 1 to 10000 μg / kg body weight / day, more preferably 10 to 1000 mg / kg body weight, / day, which may be repeated several times a day. The composition for preventing or treating hearing loss according to the present invention may also be administered in combination with a known compound having an effect of preventing, ameliorating or treating hearing loss.

Therefore, the present invention can provide a composition for preventing and treating deafness, which comprises neural cell precursor, neuron and hair cell differentiated from chorionic villi or whitetum colony-derived stem cells as an effective ingredient.

The present invention relates to a method for differentiating into a neural cell precursor, a neural cell and / or a hair cell from a chorionic membrane or a whitened umbilical cord-derived stem cell of a human placenta tissue, and a method for differentiating the precursor of the neural precursor, the neural cell and / The present invention provides a composition for the prevention or treatment of hair loss, which can be used for cell replacement therapy and regenerative medicine to treat hearing loss caused by hair cell damage, and can be used as a material for development of new drugs for hearing loss treatment In addition, there is an effect that can be widely used in basic research fields related to hearing loss treatment.

FIG. 1 is a photograph showing a microscopic observation of the morphology of cells after separating stem cells from chorionic villus or whitecostatic colony according to an embodiment of the present invention. "A" and "C" are photographs at 5 days of cultivation of chorionic villus stem cells, and "B" and "D" are photographs at 5 days of culture of stem cells derived from whiteness cord.
FIG. 2 is a photograph showing a microscopic observation of the shape of a cell according to a period of inducing differentiation of a stem cell from a chorionic membrane or a whitish umbilical cord according to an embodiment of the present invention.
FIG. 3 shows the results of FACS analysis using specific markers on the cell surface to determine whether the cells isolated from the chorion of the placenta were stem cells.
FIG. 4 shows the results of FACS analysis using specific markers on the cell surface to determine whether the cells isolated from the placenta's umbellate colony were stem cells.
FIG. 5 shows the result of performing double-labeled immunofluorescence using markers of myosin VIIA and TRPA1 to confirm the differentiation into hair cells.
FIG. 6 shows the result of performing double-labeled immunofluorescence using NF and? III-tubulin markers to confirm the differentiation into neurons.
FIG. 7 shows the result of performing double-label immunofluorescence using s100 and MBP markers to confirm the differentiation into glial cells.
8 shows a fluorescence image in a state before differentiation. The upper line shows the state before the stem cells derived from the whitening umbilical cord are differentiated, and the lower line shows the state before the stem cells derived from the chorion are differentiated. Blue indicates DAPI staining for nuclei, and green indicates staining of proliferation markers with brdU.
FIG. 9 shows the results obtained by performing RT-PCR on the differentiation and expression of neurons, hair cells, and neural precursor cells from chorionic villus or whitish umbilical cord colony-forming stem cells. &Quot; Undifferentiated cells "is a result of differentiation of stem cells derived from chorionic villi or whitish umbilical cord." Chorion MCSs " is a result of differentiation of stem cells derived from chorionic villus according to the method of the present invention. "Wharton's jelly " And the result after the stem cells derived from the colloid are differentiated according to the method of the present invention.
Fig. 10 shows the functional analysis results of differentiation-induced hair cells.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  1. Human placental chorion or Warton umbilical colloid  Isolation and culture of stem cells

The following experiments conducted in accordance with the present invention were conducted in accordance with the approval of IRB (Regulation of Clinical Research Management, KC08CIMI0344). In order to separate the chorion or farontal umbilical cord from the placenta, the placenta was first obtained from the placenta under the consent of the mother, The colloid and placenta were separated and washed with Hanks' balanced salt soultion to remove blood. The chorion or urethral colloid was then washed twice with PBS and then chopped to separate the stem cells from the chorion or whitish umbilical colloid.

Then, the cells were immersed in 0.25% trypsin EDTA and cultured in a water bath at 37 ° C and 100 rpm for 1 hour. Then, 10 ml of a culture medium containing FBS, which is an inhibitor of EDTA, was added. After 20 minutes of pipetting and centrifugation at 1000 rpm for 5 minutes, the cells were cultured in a culture medium containing 10 ng / ml of EGF for culture of stem cells Cells were cultured. After 5 days of culture, the cells in the culture broth were subjected to cell separation using trypsin to induce subculture and differentiation to obtain stem cells derived from chorionic villus or whitish umbilical cord

Example  2. Isolated chorionic villus or Warton umbilical colloid  Derived Adult stem cells  Confirm

In order to confirm that the cells derived from the placenta chorionic membrane or the whitish umbilical cord 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 HLA-DR (histocompatibility marker) antibodies were identified using a flow cytometer (FACS Caliber, Becton Dickson, San Diego, CA).

As a result, it was confirmed that the cells derived from the chorionic villus or the whitish umbilical cord were stem cells (FIGS. 2 and 3). In addition, the morphology of mononuclear cells isolated from chorionic villus or whitecellular colony was observed with increasing number of subcultures. As the number of subcultures increased, fibroblast like cell).

Example  3. Human chorionic villus or Warton umbilical colloid  Differentiation of derived stem cells into neuronal cell precursors, hair cells and neurons

The mesenchymal stem cells of the chorionic villus or the whitetum cord isolated and cultured in Example 1 were differentiated into neural precursor cells, hair cells and neurons by the following method.

3-1. Differentiation into neural cell precursors

In order to differentiate chorionic villus or whiteness cord-derived stem cells into neural cell precursors, differentiation into neural cell precursors was induced by treating nerve growth factor in the culture medium of the stem cells. In order to differentiate into neural cell precursors, Were treated with 20ng / ml EGF (Invitrogen) and 10ng / ml bFGF (basic fibroblast growth factor; Invitrogen), cultured for 21 days in the medium containing the above factors, and 10ng / ml bFGF Was added to the culture solution three times and cultured. The composition of the medium for differentiation from chorionic villus or whitened cord colony-derived stem cells to neural precursor cells is as shown in Table 1 below.

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

3-2. Differentiation into hair cells and neurons

In order to differentiate mesenchymal stem cells from chorionic villus or whitum umbilical cord by differentiation of hair cells and neurons, the compositions were changed by differentiating them from those of the neural precursor culture medium. Neurobasal medium was used as a primary culture medium, and nerve growth factors derived from glioblast-derived nerve growth factor (Invitrogen), brain-derived neurotrophic factor (Invitrogen) and neurotrophin-3 (Invitrogen) Neuronal growth factors were used, and the composition of the media used for differentiation of hair cells and neurons was as shown in Table 2 below.

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

Example  4. Immunofluorescent staining  Expression of Neuronal Cell Precursor and Hair Cells into Neurons

The neuronal cell precursors, hair cells and nerve cells obtained by differentiating from mesenchymal stem cells of the chorionic villus or whitish cord from the mesenchymal stem cells of the Example 3 were subjected to immunofluorescence staining to differentiate into neuronal cell precursors and hair cells And neuronal cells.

In order to confirm specific proliferation, BrdU was labeled on the cells for 24 hours using a BrdU (5-Bromo-2'-deoxy-uridine Labeling and Detection Kit, Roche, Indianapolis, IN) labeling. In order to investigate the differentiation into specific cells, we used glial cell markers such as GFAP, S-100, MBP, βIII-tubulin, NF, MAP2, nestin as neuronal marker, myosin VIIA, TRPA1 Were used for double staining. In addition, the expression of ctbp2 was confirmed in order to simultaneously perform functional analysis of hair cell lines. For nuclear staining, DAPI was used. Expression was counted by counting the number of cells expressed per total cell number using a fluorescence-attached microscope.

As a result, each of the stained cells was observed under a microscope. As a result, in the case of neural cell precursor differentiated from chorion or whitish umbilical cord mesenchymal stem cell, the morphology of the neural progenitor cell gradually became spherical (Fig. 2). It was confirmed that BrdU, which is a marker of cell proliferating during cell division, is a cell capable of proliferation by itself, and myosin VII and TRPA, which are markers of hair cell, are also expressed 5), neural cell markers NF and βIII-tubulin, and glial cell markers MBP and S-100 were also expressed (FIGS. 6 and 7).

Therefore, it was revealed that the chorionic villus or whitet cord-derived stem-derived stem cells according to the present invention were well differentiated into nerve cell precursors, hair cells and nerve cells.

Example  5. RT - PCR Expression of neuronal cell precursors into hair cells and nerve cells using

In Example 3, neuronal cell precursors, hair cells, and nerve cells differentiated from chorionic villi or whitened cord-derived stem cells were examined for their expression into nerve cell precursors, hair cells, and neurons by RT-PCR. For this purpose, RNA was extracted from each cell after differentiation and subjected to reverse transcription (PCR). The synthesized cDNA was denatured at 94 ° C for 5 minutes using primers of BMP4 (Bone Morphology Protein 4), BMP7, and the gene used in the immunofluorescent staining method. The DNA was reacted for 30 seconds at the binding temperature of each gene, And DNA synthesis and elongation reaction were performed for 5 minutes. GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) was used as a house keeping gene, and expression was confirmed using 2% agarose gel. The expressed genes were identified using the Image Analysis System (Bio-Rad, Hercules, Calif.).

As a result, the expression of the gene for nestin, which is a marker of stem cell, was expressed, and the expression of genes for BMP4 and BMP7, which are inner development markers, was also confirmed. Myosin VIIA and TRPA1, which are markers of hair cell, tubulin, MAP2, MBP, S-100 and GFAP genes were also expressed (Fig. 9).

Therefore, the present inventors have found that the neural precursor cells, hair cells, and neurons are well differentiated from mesenchymal stem cells of chorionic villus or whitum cord.

Example  6. Functional analysis of differentiated hair cells (FIG. 10)

Claims (11)

Comprising culturing a stem cell isolated from a human placenta chorion or warthon ' s jelly in a medium containing a nerve growth factor,
A method for differentiating a chorionic membrane or a whitened umbilical cord-derived stem cell into one selected from the group consisting of neural cell precursors, hair cells, and neurons.
2. The method of claim 1, wherein the nerve growth factor is selected from the group consisting of Epidermal Growth Factor (EGF), basic fibroblast growth factor (bFGF), Grial-derived neurotrophin factor (GDNF) Brain-derived neutrophin factor (BDNF), and neurotrophin-3 (NT-3).
The method according to claim 1, wherein the medium further comprises 1 to 3% (v / v) of B-27 supplement (B-27 supplement), 1-3 mM of L-glutamine and 0.5 to 2% ≪ / RTI >
2. The method of claim 1, wherein said culturing is performed for 15 to 26 days.
2. The method of claim 1, wherein the nerve growth factor is an epithelial growth factor or a fibroblast growth factor,
Wherein the chorionic villus or whitum umbilical cord-derived stem cells are differentiated into neural cell precursors.
6. The method according to claim 5, wherein the epithelial growth factor is contained in the medium in an amount of 5 to 15 ng / ml, and the fibroblast growth factor is contained in the medium in an amount of 15 to 25 ng / ml.
2. The method according to claim 1, wherein the nerve growth factor is at least one selected from the group consisting of neurotrophic factors derived from an adventitia-derived nerve growth factor, brain-derived neurotrophic factor and neurotrophin-
Wherein the chorionic villus or whitum umbilical cord-derived stem cells are differentiated into hair cells or neurons.
The neurotrophin-3 is added to the medium in an amount of 5 to 15 ng / ml, the neurotrophic factor in the medium is 5 to 15 ng / ml in the medium, and the neurotrophin- ng / ml. < / RTI >
A neural cell precursor differentiated from a chorionic membrane of a human placenta or a stem cell derived from a whitish umbilical cord colony, a hair cell, and a neuron,
A composition for preventing or treating hearing loss.
[Claim 11] The method according to claim 9, wherein the neural precursor cells are differentiated by culturing human placental chorionic villi or whitish cord colony-derived stem cells in a medium containing an epithelial cell growth factor or a fibroblast growth factor.
A composition for preventing or treating hearing loss.
[Claim 11] The method according to claim 9, wherein the hair cell or the neuron is selected from the group consisting of a human placenta chorionic membrane or a haematopoietic stem cell derived from at least one selected from the group consisting of a neural growth factor derived from a glioblastoma, a neurotrophin derived from a brain, Lt; RTI ID = 0.0 > nerve growth factor < / RTI >
A composition for preventing or treating hearing loss.

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