JP2012031127A - Composition including umbilical cord matrix stromal cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composition for tissue regeneration which overcomes problems on safety and ethics and which is advantageous in supply of cells.SOLUTION: A composition for transplantation using a stem cell (a umbilical cord matrix stromal cell) extracted from the umbilical cord as an active ingredient is provided. A human umbilical vein endothelial cell is used together in a preferable embodiment.

Description

  The present invention relates to a composition that is transplanted into a living body for the purpose of tissue repair or regeneration (reconstruction). Specifically, the present invention relates to a transplant composition using stem cells derived from the umbilical cord and its use (treatment using the composition, etc.).

  Although organ transplantation and cell transplantation using embryonic stem cells (ES cells) have been studied for congenital neonatal diseases (see, for example, Patent Document 1), organ transplantation is an organ shortage problem, and ES cells are safety and ethics. Have problems. On the other hand, cell transplantation treatment using bone marrow-derived autologous mesenchymal stem cells is being applied, but collecting stem cells from bone marrow is accompanied by severe biological invasion. In particular, it is practically impossible to collect cells from the bone marrow of newborns with congenital diseases. There are also attempts to use cells derived from human umbilical cord blood for regenerative medicine (see, for example, Patent Document 2 and Non-Patent Document 1), but the presence of stem cells in umbilical cord blood is very low, and the stability of stem cells Supply is difficult.

JP-T-2005-521402 JP 2008-179642 A

Mariane secco et al., Stem cells 2008; 26: 146-150

  Therefore, an object of the present invention is to provide a composition useful for tissue repair or regeneration, which can overcome safety and ethical problems and is advantageous in terms of cell supply, and its use.

  Under the above problems, the present inventors have focused on “umbilical cords that are disposed of as medical waste after delivery”. The umbilical cord with a total length of about 30-70cm is a fetal tissue, and a large amount of umbilical cord-derived mesenchymal stem cells from "Wharton's jelly" around the umbilical blood vessels delivered by vaginal delivery and caesarean section (Umbilical cord matrix stromal cells: UCMSC) can be collected, and at the same time, umbilical vein endothelial cells (HUVEC) useful for angiogenesis in tissue regeneration can be collected from the umbilical vein.

  The inventors first decided to analyze various characteristics of UCMSC. As a result, UCMSC showed significantly higher proliferation ability than bone marrow-derived mesenchymal stem cells (Bone Marrow Mesenchymal Stem Cell: BMMSC). Further, as features of UCMSC, CD73 positive, CD90 positive, CD105 positive, CD11b negative, CD34 negative, CD45 negative, and HLA-DR negative were confirmed. On the other hand, it has been shown that it has the ability to differentiate into bone cells, chondrocytes and adipocytes, and it is also possible to differentiate into nerve cells. In further studies, it was found that the induction of differentiation into bone cells can be promoted by adding bone morphogenetic protein 2 (Bone Morphogenetic Protein 2: BMP-2) to the medium. In addition, it was suggested that co-culture with HUVEC or culture under hypoxic conditions enhances the ability of UCMSC to differentiate into bone. On the other hand, as a result of transplantation experiments, it was found that UCMSC is extremely useful as a cell source for bone regeneration and wound healing, and that regeneration or treatment effect is improved by using HUVEC together.

As described above, as a result of intensive studies by the inventors of the present application, it was found that UCMSC was extremely excellent as a cell source for regenerative medicine, and a lot of useful information was brought about when UCMSC was applied clinically. It was. Based on the above results, the following inventions are provided.
[1] A composition for transplantation comprising umbilical cord-derived mesenchymal stem cells.
[2] The composition for transplantation according to [1], wherein the umbilical cord-derived mesenchymal stem cells are derived from Wharton's jelly.
[3] The composition for transplantation according to [1] or [2], which is used for treatment of neonatal periventricular leukomalacia, congenital heart disease or congenital cleft lip and palate.
[4] The composition for transplantation according to [1] or [2], wherein the umbilical cord-derived mesenchymal stem cells are undifferentiated umbilical cord-derived mesenchymal stem cells not subjected to differentiation induction treatment after collection. object.
[5] The transplant composition according to [4], wherein the umbilical cord-derived mesenchymal stem cells are CD73 positive, CD90 positive, CD105 positive, CD11b negative, CD34 negative, CD45 negative, and HLA-DR negative.
[6] The composition for transplantation according to [4] or [5], which is for regeneration of skin tissue or nerve tissue.
[7] The composition for transplantation according to [1] or [2], wherein the umbilical cord-derived mesenchymal stem cell is a differentiation-inducing umbilical cord-derived mesenchymal stem cell that has been induced to differentiate into a specific cell lineage. object.
[8] The composition for transplantation according to [7], which is for regeneration of bone tissue, cartilage tissue, nerve tissue, muscle tissue, or vascular tissue.
[9] The transplant according to [7], wherein the differentiation-inducing umbilical cord-derived mesenchymal stem cell has alkaline phosphatase activity and is positive for type I collagen, RUNX2 and osteocalcin, and is used for bone tissue regeneration. Composition.
[10] The composition for transplantation according to [7], wherein the differentiation-inducing umbilical cord-derived mesenchymal stem cells are cells in which alkaline phosphatase activity is enhanced by stimulation with bone morphogenetic protein 2 (BMP-2).
[11] The transplant composition according to [7], wherein the differentiation-inducing umbilical cord-derived mesenchymal stem cells are cells in which alkaline phosphatase activity is enhanced by co-culture with umbilical vein endothelial cells.
[12] The composition for transplantation according to [7], wherein the differentiation-inducing umbilical cord-derived mesenchymal stem cells are cells in which alkaline phosphatase activity is enhanced by culturing in a hypoxic environment.
[13] The composition for transplant according to [12], wherein the culture in a hypoxic environment is a culture using a medium to which deferoxamine is added.
[14] The composition for transplantation according to any one of [1] to [13], which is a combination of umbilical vein endothelial cells.
[15] The transplant composition according to [14], comprising umbilical cord-derived mesenchymal stem cells and umbilical vein endothelial cells.
[16] The transplant composition according to [14], which is a kit comprising a first component containing umbilical cord-derived mesenchymal stem cells and a second component containing umbilical vein endothelial cells. object.
[17] The composition for transplantation according to [14], comprising umbilical cord-derived mesenchymal stem cells, and umbilical vein endothelial cells are transplanted together at the time of transplantation.
[18] The composition for transplantation according to any one of [14] to [17], wherein the donor of umbilical cord-derived mesenchymal cells and the donor of umbilical vein endothelial cells are the same.
[19] A tissue repair or regeneration method comprising injecting, embedding, filling, or applying the transplant composition according to any one of [1] to [18] to a target site .
[20] A method for preparing a cell having enhanced alkaline phosphatase activity, comprising co-culturing umbilical cord-derived mesenchymal stem cells and umbilical vein endothelial cells.
[21] A method for preparing a cell having enhanced alkaline phosphatase activity, comprising a step of culturing umbilical cord-derived mesenchymal stem cells in a hypoxic environment.

FIG. 1 is a graph showing the proliferation ability of UCMSC. The proliferation rate was compared between UCMSC derived from umbilical cord obtained by vaginal delivery (●), UCMSC derived from umbilical cord obtained by caesarean section (▲), and bone marrow mesenchymal stem cells (■). * There is a significant difference. FIG. 2 shows the results of analysis of cell surface markers of UCMSC. A flow cytometry chart for each surface antigen is shown. Fig. 3 shows ULSISC-alizarin red-stained image of UCMSC after induction of differentiation into bone cells (lower left), UCCSC Alcian blue-stained image after induction of differentiation into chondrocytes, and after induction of differentiation into adipocytes. Oil-red-o stained image of UCMSC (bottom center), stained image of UCMSC after differentiation induction into neurons (upper right: anti-nestin antibody / DAPI staining, lower right: anti-βIII-tubulin antibody / DAPI) . The upper left is a microscope image of UCMSC after expansion culture. FIG. 4 shows the result of analyzing the characteristics of umbilical cord-derived HUVEC. The upper left is a microscopic image of HUVEC after expanded culture, and the upper middle (anti-vWF antibody / DAPI) and the upper right (anti-CD31 antibody / DAPI) are fluorescent immunostained images of HUVEC. The lower row is the result of flow cytometry. FIG. 5 is a graph (upper) showing the alkaline phosphatase activity of UCMSC cultured in a medium supplemented with BMP-2, and the results of analyzing gene expression of osteoprogenitor cell lineage markers (lower). * There is a significant difference. FIG. 6 is a graph comparing the alkaline phosphatase activity (day 14) between the group cultured with UCMSC alone (UCMSC) and the group co-cultured with UUVSC (UCMSC & HUVEC). FIG. 7 shows UCMSC (Dex (+)) cultured in a normal bone differentiation-inducing medium and UCMSC (Dex (+)-DFO (+)) cultured by adding BMP-2 to a normal bone differentiation-inducing medium. A graph comparing the alkaline phosphatase activity (day 7) (top) and the results of analyzing the gene expression of hypoxia response factors and angiogenic factors (bottom). As a negative control, UCMSC (Dex (−)) cultured in a bone differentiation induction medium without dexamethasone was used. FIG. 8 shows the results of UCMSC transplantation experiment using an ectopic ossification study model (hematoxylin-eosin stained image 6 weeks after transplantation). A pocket was formed subcutaneously in the back of nude mice, and cell preparations were transplanted. Six weeks later, histological evaluation was performed. The left is the SHAM group, the center is the UMSSC and hydroxyapatite mixture transplant group, and the right is the UCMSC, HUVEC and hydroxyapatite mixture transplant group. FIG. 9 shows the results of UCMSC transplantation experiment using the wound healing study model (graph of wound reduction rate). Cells suspended in type I collagen gel were transplanted into nude mice in which the entire epidermis layer was excised with a biopsy punch and wounds were made. The wound reduction rate was compared between the group using only UCMSC (▲) and the group using UCMSC and HUVEC together (●).

(Composition using umbilical cord-derived stem cells)
The first aspect of the present invention relates to a composition for transplantation using umbilical cord-derived stem cells. As used herein, the term “transplant composition” refers to a composition prepared ex vivo, which is used for the purpose of repairing or regenerating tissues or organs in which a defect such as a defect, malformation, or dysfunction is recognized. The one to be transplanted. The composition of the present invention is used for, for example, repair or regeneration of bone tissue, cartilage tissue, adipose tissue, nerve tissue, skin tissue, muscle tissue, or vascular tissue. The composition of the present invention requires various post-surgical operations for treatment of various diseases in which tissue regeneration has a therapeutic effect, such as neonatal periventricular leukomalacia, a congenital neurological disease, or congenital heart disease. Treatment of things and congenital cleft lip and palate, treatment of bone defects and bone deformities, various neurological diseases (traumatic diseases such as spinal cord injury, peripheral nerve palsy, amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, progression Neurodegenerative diseases such as paralysis of nuclei, Huntington's disease, multiple system atrophy, spinocerebellar degeneration, cerebral ischemia, cerebral infarction due to intracerebral hemorrhage, etc., retina with neuronal damage Disease), wound healing (promoting wound healing, keloid prevention, scar prevention, and / or skin quality improvement) and the like.

  The composition of the present invention comprises umbilical cord-derived mesenchymal stem cells (hereinafter sometimes referred to as “UCMSC” for convenience of explanation). The umbilical cord is a tubular tissue connecting the fetus and the placenta, and contains two arteries and one vein. Surrounding these blood vessels is a tissue called Wharton's jelly. The cells constituting the composition of the present invention are preferably derived from the tissue.

  An example of a method for collecting and preparing UCMSC is shown below. In this collection / preparation method, (1) umbilical cord collection, (2) enzyme treatment, (3) cell culture, and (4) cell recovery are sequentially performed.

(1) Umbilical cord collection Collect the umbilical cord delivered by vaginal delivery and cesarean section. After removing cord blood, sterilization is performed. Popidone iodine or the like can be used for aseptic processing. The umbilical cord is opened with a scalpel and the umbilical vein and umbilical artery are separated with the surrounding Wharton's jelly.

(2) Enzyme treatment After the separated and collected tissue is treated with collagenase, the umbilical artery and vein are removed. Subsequently, the cells (UCMSC) are recovered by centrifugation for 5 minutes (5000 rpm).

(3) Cell culture The cells are suspended in a 4 cc mesenchymal stem cell medium and seeded in an adherent cell culture dish having a diameter of 6 cm. Incubate in an incubator adjusted to 37 ° C with 5% CO ₂ . Cells are treated with 0.05% trypsin EDTA for 5 minutes at 37 ° C when subconfluent (cells occupy about 70% of the surface of the culture vessel) or confluent. The UCMSC exfoliated from the dish is seeded in an adherent cell culture dish having a diameter of 10 cm and expanded. Subculturing may be repeated. For example, the subculture is performed 1 to 8 times to grow to the required number of cells (for example, about 1 × 10 ⁷ cells / ml). After the above culture, the cells may be collected and stored (storage conditions are, for example, -198 ° C.). When stored in this way and applied to the treatment of future diseases such as bone, cartilage, and nerve (especially intractable diseases), safe and minimally invasive regenerative medicine using autologous cells can be provided. Note that cells collected from various donors may be stored in the form of a UCMSC bank.

(4) Cell recovery Next, cells are recovered. The cells can be collected by centrifuging after detaching the cells from the culture vessel by trypsin treatment or the like. The composition of the present invention is prepared using the cells thus recovered.

  In one embodiment of the present invention, UCMSC prepared by the preparation method as described above is used in the composition of the present invention without inducing differentiation into specific cells. That is, UCMSC that is not differentiated after collection (also referred to herein as “undifferentiated umbilical cord-derived mesenchymal stem cells” or “undifferentiated UCMSC”) is used as an active ingredient. The composition of this embodiment is effective for wound healing (see Examples described later) and nerve tissue regeneration, for example. UCMSCs used in this embodiment are CD73 positive, CD90 positive, CD105 positive, CD11b negative, CD34 negative, CD45 negative, HLA-DR negative. The presence or absence of expression of these cell surface markers may be examined by a conventional method. For example, the presence or absence of expression of a specific cell surface marker can be easily examined by using flow cytometry or fluorescent immunohistochemical staining. For example, FACS Calibur (Becton Dickinson) can be used for flow cytometry analysis. For example, a confocal laser microscope A1Rsi (Nikon Corporation) can be used for analysis of multiple stained specimens after nuclear staining with DAPI.

  In another embodiment, the prepared UCMSC is used for the composition of the present invention after being induced to differentiate into a specific cell lineage. The UCMSC subjected to differentiation induction treatment is referred to herein as “differentiation-inducing umbilical cord-derived mesenchymal stem cells” or “differentiation-inducing UCMSC”. Depending on the purpose of use, specific cells (for example, bone cells (osteoblasts, bone cells, osteoclasts, chondrocytes, etc.), nervous cells (various central nerve cells such as dopamine-producing cells, peripheral nerve cells, astrocytic cells) Differentiation-inducing UCMSCs that have been induced to differentiate into cells, oligodendrocytes, Schwann cells), myoblasts, myocytes, cardiomyocytes, adipocytes).

  For example, differentiation-inducing UCMSCs that have been induced to differentiate into bone cells are effective in the regeneration of bone tissue and cartilage tissue, and are suitable for the treatment of congenital cleft lip and palate, bone defects and bone deformation. Similarly, differentiation-inducing UCMSCs that have been induced to differentiate into nervous system cells are effective in the regeneration of nerve tissue, and traumatic diseases such as chronic spinal cord injury, chronic peripheral nerve paralysis, and chronic cerebral infarction (preferably mature neurons and Oligodendrocyte Schwann cells differentiated cells), amyotrophic lateral sclerosis (preferably spinal cord, brainstem motor neurons differentiated), Parkinson's disease (preferably dopamine) Suitable for the treatment of spinocerebellar degeneration (preferably using cells induced to differentiate into cerebellar Purkinje cells or granule cells).

Differentiation induction into specific cells can be performed by a conventional method. As an example, a typical technique for inducing differentiation into osteoblasts or bone cells includes three additives in the culture medium: dexamethasone (Dex) and β-glycerophosphate sodium (β-GP). , And by adding L-ascorbic acid diphosphate (AsAP) (or by replacing with a medium containing these additives (osteoinduction medium)), it promotes differentiation into bone cells. The addition amount of these additives is, for example, about 10 ⁻⁷ M for dexamethasone, about 10 mM for β-glycerophosphate sodium, and about 0.2 mM for L-ascorbic acid diphosphate. Preferably, osteogenic protein 2 (Bone Morphogenetic Protein 2; BMP-2) is also used in combination to promote differentiation into bone cells. The concentration of BMP-2 added is preferably 50 ng / ml to 100 ng / ml. Medium exchange is performed appropriately during differentiation induction. For example, the medium is changed once every three days. The culture for inducing differentiation is continued, for example, for 1 day to 14 days.

On the other hand, a method comprising the following two steps can be used for inducing differentiation into a dopaminergic neuron that is a kind of nerve cell. In the first step, using a dish coated with poly-L-lysine, for example, 2 to 3 dental pulp stem cells were cultured in DMED medium containing 12.5 U / ml Nystatin, N2 supplement, 20 ng / ml bFGF and 20 ng / ml EGF. Incubate for days. Through this step, dental pulp stem cells are induced to differentiate into neural stem cells. In the second step, the cells after the first step are cultured for 6 to 7 days in Neurobasal ^™ medium containing, for example, B27 supplement, 1 mM db-cAMP, 0.5 mM IBMX, 200 μM ascorbic acid and 50 ng / ml BDNF. The induced dopaminergic neurons can be confirmed by immunostaining using an antibody against tyrosine hydroxylase. In addition to the above method, a method of culturing in a floating aggregation culture system after culturing in the presence of bFGF (Studer, L. et al .: Nat. Neurosci., 1: 290-295, 1998), bFGF and glial cell line Method of culturing in the presence of culture supernatant (Daadi, MM and Weiss, SJ: Neuroscience, 19: 4484-4497, 1999.), method using FGF8, Shh, bFGF, ascorbic acid, etc. (Lee, SH et al .: Nat. Biotechnol., 18: 675-679, 2000.), neural stem cells, including methods of co-culture with bone marrow stromal cells (Kawasaki, H. et al .: Neuron, 28: 31-40, 2000.) Alternatively, various methods reported as methods for inducing differentiation of embryonic stem cells into dopaminergic neurons may be used after appropriately modifying as necessary.

  In addition, a method comprising the following two steps can be used to induce differentiation into astrocytes. In the first step, dental pulp stem cells are cultured for 4 days in a DMEM / F12 medium containing, for example, N2 supplement and 10 ng / ml bFGF, using a dish in which poly-L-ornithine and fibronectin are double-coated. In the second step, the cells are further cultured for 3 days in a medium supplemented with 80 ng / ml LIF and 80 ng / ml BMP2. Differentiated astrocytes can be confirmed by immunostaining using an antibody against GFAP.

  For the induction of differentiation into oligodendrocytes, the following two-step method can be used. Similar to the induction of differentiation into astrocytes, in the first step, a dish with a double coat of poly-L-ornithine and fibronectin is used, for example in DMEM / F12 medium containing N2 supplement, 10 ng / ml bFGF and 0.5% FCS. Pulp stem cells are cultured for 4 days. This process induces dental pulp stem cells into oligodendrocyte progenitor cells. In the subsequent second step, the cells are cultured for 4 days in a DMEM / F12 medium containing 20 ng / ml T3 (Triiodothyronine), 20 ng / ml T4 (Thyroxine) and N2 supplement. Differentiated oligodendrocytes can be confirmed using an antibody against O4.

  As shown in Examples described later, when UCMSC was co-cultured with HUVEC, surprisingly, a significant increase in alkaline phosphatase (ALP) activity, which is an index of differentiation into bone cells, was observed. This suggests that co-culture with HUVEC enhances the alkaline phosphatase activity of UCMSC, in other words, it can enhance the ability of UCMSC to differentiate into bone. Based on this finding, in one embodiment of the present invention, cells in which alkaline phosphatase activity is enhanced by co-culture with HUVEC are used. According to the composition of this aspect, a higher bone tissue regeneration effect can be expected. The above findings mean that cells with enhanced alkaline phosphatase activity can be obtained if UCMSC is co-cultured with HUVEC, which itself has important value.

  Co-culture with HUVEC can be carried out alone or with the above differentiation induction procedure (ie, dexamethasone, β-glycerophosphate sodium and L-ascorbate diphosphate, or BMP-2 in addition to these three components) In combination with a bone cell-derived differentiation operation using a medium or the like to which is added. In the latter case (in combination with the differentiation inducing operation described above), for example, a culture step in which co-culture with HUVEC is performed after the differentiation inducing operation, or the differentiation inducing operation is performed after the co-culture with HUVEC. Any of the above-described culture process and the above-described culture process in which the differentiation induction operation is performed in the presence of HUVEC may be employed.

  On the other hand, by culturing in a medium supplemented with deferoxamine (DFO), which is known as a reagent that forms a pseudo-hypoxic state, surprisingly, there is a significant increase in alkaline phosphatase (ALP) activity, which is an indicator of differentiation into bone cells. (See Examples below). This suggests that the alkaline phosphatase activity of UCMSC is enhanced by culturing in a hypoxic environment, in other words, the ability of UCMSC to differentiate into bone can be enhanced. Based on this finding, in one embodiment of the present invention, cells whose alkaline phosphatase activity is enhanced by culturing in a hypoxic environment are used. According to the composition of this aspect, a higher bone tissue regeneration effect can be expected. The above findings mean that cells with enhanced alkaline phosphatase activity can be obtained by culturing UCMSC in a hypoxic environment, which itself has important value.

  In this specification, the term “under a hypoxic environment” means that a cell has the same stimuli and effects as those in a hypoxic state, although the oxygen concentration is not low in a low oxygen atmosphere (that is, in an environment where oxygen is actually low). It includes under the environment to be given (that is, under a hypoxic environment). Here, “low oxygen” means an oxygen concentration lower than the normal oxygen concentration (21%). The concentration of oxygen in the low oxygen state is preferably 10% or less, more preferably 5% or less, and still more preferably 2% or less. A minimum oxygen concentration is necessary to maintain the survival of the cells subjected to culture. Therefore, the lower limit value of the oxygen concentration is, for example, 0.01%, preferably 0.1%, more preferably 0.5%, and still more preferably 1.0%.

The pseudo-hypoxic environment can be formed by a hypoxia-inducing agent such as deferoxamine (DFO), CoCl ₂ , and picolinic acid. Preferably, hypoxia is formed by adding deferoxamine (DFO) to the medium.

  Cultivation in a hypoxic environment can be carried out alone or with the above-described differentiation-inducing operation (ie, dexamethasone, β-glycerophosphate sodium and L-ascorbic acid diphosphate added medium, or BMP in addition to these three components) (Operation for inducing differentiation into bone cells using a medium supplemented with -2) and / or co-culture with HUVEC. If the specific example of the culture | cultivation process in the latter case (in combination with said differentiation induction operation and / or co-culture with HUVEC) is shown, the culture process of performing culture | cultivation in a hypoxic environment after said differentiation induction operation. A culture process in which the above differentiation induction operation is performed after culture in a hypoxic environment, a culture process in which the above differentiation induction operation is performed in a low oxygen environment, and a culture with HUVEC after culture in a low oxygen environment. A culture step in which culture is performed, followed by the differentiation induction operation described above, and a culture step in which the differentiation induction operation is performed after culture in a hypoxic environment and then co-culture with HUVEC is performed. The above findings mean that cells with enhanced alkaline phosphatase activity of UCMSC can be obtained when co-cultured with HUVEC, which itself has important value.

  In one embodiment of the present invention, umbilical cord-derived mesenchymal stem cells (UCMSC) and umbilical vein endothelial cells (HUVEC) are used in combination. In this specification, “Umbilical cord-derived mesenchymal stem cells (UCMSC) and umbilical vein endothelial cells (HUVEC) are used in combination” or “Umbilical cord-derived mesenchymal stem cells (UCMSC) and umbilical vein endothelial cells (HUVEC) are combined” "Is synonymous with the combined use of umbilical cord-derived mesenchymal stem cells (UCMSC) and umbilical vein endothelial cells (HUVEC)." A specific mode of combined use will be described later.

  Preferably, the UCMSC donor and the HUVEC donor are the same. In other words, it is preferable to use a combination of UCMSC and HUVEC obtained from a specific umbilical cord. This is because a better interaction can be expected, and an application in which the donor of both cells and the recipient receiving the transplant are identical (that is, complete autotransplantation) becomes possible. HUVEC has the characteristics of CD31 positive as a mature endothelial cell marker, CD34 positive as an endothelial progenitor cell marker, and CD45 negative as a blood cell marker (see Examples below).

  As shown in the examples described later, as a result of the study by the present inventors, by using UCMSC and HUVEC in combination, surprisingly, a significant increase in alkaline phosphatase (ALP) activity, which is an indicator of differentiation into bone cells, is achieved. An increase was observed. This fact indicates that the combined use of UCMSC and HUVEC is particularly effective for the purpose of bone tissue regeneration, and that such an application form has an advantageous effect.

  On the other hand, when UCMSC and HUVEC were used in combination, it was revealed that a high therapeutic effect on wounds was achieved. Therefore, the combined use of UCMSC and HUVEC is suitable for application to wound healing and brings about an excellent therapeutic effect.

  In the case of this embodiment in which UCMSC and HUVEC are used in combination, for example, the transplant composition of the present invention is provided as a combined preparation of UCMSC (undifferentiated UCMSC or differentiation-inducing UCMSC) and HUVEC. In such a compounded composition for transplantation, HUVEC prepared in advance as in the case of UCMSC prepared in advance may be mixed using physiological saline or a suitable buffer (for example, phosphate buffer) as a solvent.

  On the other hand, for example, the composition for transplantation of the present invention can be provided in the form of a kit comprising a first component containing UCMSC and a second component containing HUVEC. In this case, both elements are transplanted to the recipient at the same time or at a predetermined time interval. Preferably, both elements will be implanted simultaneously. This is to maximize the effect of the interaction between UCMSC and HUVEC. “Simultaneous” here does not require strict simultaneity. Therefore, of course, if both elements are transplanted to the subject after mixing both elements, such as when the transplantation of both elements is performed under conditions that do not have a time difference, after transplanting one of the elements, the other is quickly transplanted. The case where the transplantation is performed under conditions without a substantial time difference is also included in the concept of “simultaneous” herein. On the other hand, when transplanting the other with a predetermined time difference after transplanting one, it is preferable to set the time difference as short as possible so that the interaction between the two elements is excellent. For example, the other is transplanted within 3 hours, preferably within 1 hour, more preferably within 30 minutes after one transplant.

  A composition for transplantation containing UCMSC may be used, and HUVEC may be transplanted together at the time of transplantation. The timing of transplantation of the transplant composition and HUVEC in this case is the same as in the case of the kit form described above. That is, both are preferably transplanted at the same time, but they may be transplanted at a predetermined time difference. In contrast to the above-described embodiment, a composition for transplantation containing HUVEC may be used, and UCMSC may be transplanted together at the time of transplantation. The timing of transplantation in this case conforms to the case of the above embodiment.

For example, 0.5 × 10 ⁶ to 2 HUVECs may be included in the transplantation composition so that a desired amount of regenerative effect is exhibited, such as 0.5 × 10 ⁶ to 2 × 10 ⁷ UCMSC as a single dose. × 10 ⁷ cells (one dose) are used. The amount of UCMSC and HUVEC can be appropriately adjusted in consideration of the purpose of use, application site (particularly the size of the defect or the state of the affected area), the state of the cells, and the like.

  In one aspect, the composition of the present invention is prepared in a gel form for reasons such as improved operability and therapeutic effects. The term “gel” in the present specification refers to a state having an appropriate viscosity and high retention at the transplanted part, such as fibrin gel or fibrin glue used for medical purposes. For example, a gel-like composition is formed by adding a gelling agent or a thickener, or adding fibrinogen and thrombin.

It does not preclude the additional use of other ingredients, provided that the therapeutic effect expected of the composition of the present invention is maintained. Ingredients that can be additionally used in the present invention, including materials for preparing gels, are listed below.
(1) Inorganic bioabsorbable material and organic bioabsorbable material The type of inorganic bioabsorbable material is not particularly limited, but β-tricalcium phosphate (“β-TCP), α-tricalcium phosphate ( α-TCP), tetracalcium phosphate, octacalcium phosphate, and amorphous calcium phosphate can be used.These materials can be used alone or of course. Two or more selected from the above may be used in combination, preferably either β-TCP or α-TCP, or a combination thereof in any proportion, more preferably β-TCP is inorganic bioabsorbable. Used as a functional material.

  The inorganic bioabsorbable material can be obtained by a known method. Moreover, a commercially available inorganic bioabsorbable material can also be used. As β-TCP, for example, those manufactured by Olympus Optical Co., Ltd. can be used.

  The inorganic bioabsorbable material is preferably in the form of a powder having a particle diameter such that the composition of the present invention becomes fluid when used. The powdered inorganic bioabsorbable material can be prepared by crushing and pulverizing the inorganic bioabsorbable material processed to an appropriate size until a desired particle diameter is obtained. The average particle diameter of the inorganic bioabsorbable material is preferably 0.5 μm to 50 μm. More preferably, an inorganic bioabsorbable material having an average particle size of 0.5 μm to 10 μm is used. Even more preferably, an inorganic bioabsorbable material having an average particle diameter of 1 μm to 5 μm is used. It is also possible to use a combination of a plurality of types of inorganic bioabsorbable materials having different particle diameters. The inorganic bioabsorbable material is preferably contained in an amount of 30% to 75% by weight based on the entire composition of the present invention.

  In addition, the fluidity | liquidity of the composition of this invention can be adjusted with the particle diameter and content rate of an inorganic type bioabsorbable material, and desired fluidity | liquidity can be obtained by adjusting both suitably. Moreover, when adding the below-mentioned thickener, fluidity | liquidity can be adjusted also with the addition amount of a thickener.

  As the organic bioabsorbable material, hyaluronic acid, collagen, fibrinogen (for example, Bolheel (registered trademark)) or the like can be used.

(2) Gelling material It is preferable to use a gelling material having high biocompatibility, and hyaluronic acid, collagen, fibrin glue, or the like can be used. Various types of hyaluronic acid and collagen can be selected and used, but it is preferable to employ one suitable for the application purpose (application tissue) of the composition of the present invention. The collagen used is preferably soluble (acid-soluble collagen, alkali-soluble collagen, enzyme-soluble collagen, etc.).

(3) Solvent The composition of the present invention may contain an aqueous solvent. As the aqueous solvent, sterilized water, physiological saline, a buffer solution such as a phosphate solution, or the like can be used. The prepared cells can be suspended in physiological saline or PBS (phosphate buffered physiological saline) to obtain the composition of the present invention (not containing other components) and applied to the affected area.

(4) Others In addition to the above components, the composition of the present invention comprises a carrier, excipient, disintegrant, buffer, emulsifier, suspension, soothing agent, stabilizer, preservative, preservative, cell protection. Agents (eg, dimethyl sulfoxide (DMSO) and serum albumin), antibiotics, pH adjusters, various components for the purpose of cell activation, proliferation or differentiation induction (vitamins, cytokines, growth factors, steroids, etc.) ) May be included.

(Method of applying)
The composition of the present invention is transplanted to a tissue defect or a damaged site by, for example, injection, embedding, filling, or application. If it is prepared in the form of a gel having appropriate fluidity, it can be applied by a simple procedure such as filling, pouring or coating. Moreover, if it is a gel, it can be easily inserted into the application site using an injection needle or the like (it can also be applied without opening the affected area). Moreover, it does not need to shape | mold beforehand according to the shape of a tissue defect | deletion part, and the versatility is high.

1. Method for simultaneous separation and preparation of UCMSC and HUVEC from umbilical cord tissue
a. Separation of Vascular Endothelial Cells from Umbilical Vein The umbilical cord can be the umbilical cord delivered by vaginal delivery and cesarean section. The umbilical cord of 10 cm to 20 cm from which the umbilical cord blood has been removed is immersed in a 10% isodine (Isodine (registered trademark) solution 10%, Meiji Seika) solution for 1 minute for aseptic treatment. Then wash twice with PBS. After confirming the umbilical vein of both umbilical cord fragments, a washing needle is inserted and fixed with a clamp. Attach a 20 ml syringe to the washing needle and wash 2 to 3 times with 20 ml of PBS until no blood remains. One piece is clamped and 5 ml of 0.05% trypsin is injected into the umbilical vein. Incubate for 15-20 minutes in a 500 ml beaker with PBS at 37 ° C. with the umbilical cord in a U shape. Then, lightly massage the umbilical cord. From one washing needle, inject 10 ml of basic medium (Dulbecco's modified Eagle's medium containing 10% bovine serum / antibiotics) and collect into a 50 cc conical tube. Centrifuge at 200 xg for 10 minutes. endothelial cells were resuspended in a dedicated media (EBM-2, Lonza Inc.), and cultured in 6cm of adherent cells .5% CO to seed culture dish ₂ · 37 ° C. to adjust incubator. after incubation, 80 If% confluent, treat with 0.05% trypsin / EDTA for 5 minutes at 37 ° C. Seed vascular endothelial cells detached from the dish in a 10 cm adherent cell culture dish and perform expansion culture.

b. Separation of UCMSC from Wharton's jelly around umbilical arteriovenous blood vessel The umbilical cord from which vascular endothelial cells have been collected is immersed again in 10% isodine solution for 1 minute to perform aseptic treatment. Then wash twice with PBS. After opening the umbilical cord with a scalpel, reveal one umbilical vein and two umbilical arteries. Separate the umbilical vein and umbilical artery with the surrounding Wharton's jelly. To prevent contamination with vascular endothelial cells, the stump is ligated with silk thread. Three ligated umbilical arteries and veins are treated with 1 mg / ml collagenase at 37 ° C for 3 hours. Remove the umbilical arteriovenous and collect UCMSC by centrifugation for 5 minutes (5000 rpm). The cell pellet is resuspended in 4cc mesenchymal stem cell-dedicated medium (MSCBM, Lonza) and seeded in a 6cm adherent cell culture dish. Culture in an incubator adjusted to 5% CO ₂ · 37 ° C. When 80% confluent after incubation, treat with 0.05% trypsin / EDTA for 5 minutes at 37 ° C. UCMSC exfoliated from the dish is seeded on a 10 cm adherent cell culture dish and expanded.

<Result>
Regardless of whether it was delivered by vaginal delivery or cesarean section, UCMSCs that had undergone primary culture showed the appearance of colonies in 3-5 days. On the 15th to 21st days from the start of the culture, the cells became confluent, and expansion culture was possible by subculture (FIG. 3). HUVEC that had undergone primary culture became confluent about 5 days after the start, and expansion culture was possible by passage (FIG. 4).

  In the past, only the umbilical cord delivered aseptically by caesarean section was indicated for cell collection. However, the umbilical cord was aseptically soaked in 10% isodine (Isodin (registered trademark) 10%, Meiji Seika) for 1 minute. By adding a new process, we found a method to collect cells without bacterial infection from specimens delivered by normal vaginal delivery. It has been experimentally confirmed that even if aseptic processing is performed by this method, it does not affect the proliferation ability and differentiation ability of the collected stem cells.

2. Characteristic analysis of UCMSC <Method>
a.Proliferation test
To investigate the cell growth ability of UCMSC. For comparison, human bone marrow mesenchymal stem cells (BMMSC, Lonza) are used. 2 × 10 ⁴ cells are seeded in each well of a 12-well adherent cell culture dish. MSCBM is used as the medium, and the medium is changed 3 days after sowing. Treated with 0.05% trypsin / EDTA for 5 minutes at 37 ° C. 1 day, 3 days, and 6 days after seeding, the cells detached from the dish are collected, stained with trypan blue, and the number of viable cells is counted.

b. Flow cytometry Cell surface markers are analyzed by flow cytometry. FACS Calibur (Becton Dickinson) was used for the analysis.

c. Pluripotency analysis
c-1. Confirmation of differentiation potential into bone cells Cells are seeded on adherent cell culture dishes at 3.1 × 10 ³ cells / cm ² . Bone differentiation induction medium ( ^10-7 M dexamethasone, 10 mM β-glycerophosphate, 0.2 mM L-ascorbic acid-2-phosphate MSCBM) Incubate at During differentiation induction, the medium is changed once every 2-3 days. Differentiation is induced for about 4 weeks, and the induced osteoblasts are stained with alizarin red and confirmed along with calcification.

c-2. Confirmation of differentiation potential into chondrocytes
2.5 × 10 ⁵ cells in a 15 ml polypropylene tube 50 μg / ml L-ascorbic acid-2-phosphate, 40 μg / ml L-proline, 100 μg / ml pyruvin Sodium pyruvate, 0.1μM dexamethasone, 6.25μg / ml insulin, 6.25μg / ml transferrin, 6.25μg / ml selenous acid, 10ng / ml human TGF-β3 Inoculate with MSCBM supplemented with (human TGF-β3), centrifuge for 5 minutes (5000 rpm), and culture in pellet form. During differentiation induction, medium is changed once every 2-3 days, and pellet culture is performed for about 4 weeks. The induced cartilage tissue is confirmed by preparing a tissue section and staining with Alcian blue.

c-3. Confirmation of differentiation potential into adipocytes Cells are seeded in adherent cell culture dishes at 2.1 × 10 ⁴ cells / cm ² and cultured until confluent. Incubate in MSCBM supplemented with 1 μM dexamethasone, 1 μg / ml insulin, 0.5 mM 3-isobutyl-1-methylxanthine for about 3 weeks. Induced adipocytes are confirmed by Oil-red-o staining.
c-4. Confirmation of differentiation ability into neurons In order to induce differentiation into neurons, culture for 12 days in a medium supplemented with 0.5% BSA, 25μg / ml FGF8, 25μg / ml SHH, 25μg / ml b-FGF . Induced nerve cells are confirmed by immunostaining using antibodies against Nestin and βIII-tubulin.

<Result>
a. Proliferation test UCMSC showed good proliferative ability regardless of vaginal delivery or cesarean section. UCMSC has a high proliferation ability and is significantly higher than BMMSC. It can also be seen that aseptic treatment with 10% isodine (Isodine (registered trademark) solution 10%, Meiji Seika) solution does not affect cell growth (FIG. 1).

b. Flow cytometry
UCMSC was found to be CD73 positive, CD90 positive, CD105 positive, CD11b negative, CD34 negative, CD45 negative, and HLA-DR negative (FIG. 2).

c. Pluripotency analysis
UCMSC cultured for 28 days in a bone differentiation-inducing medium was stained with alizarin red. As a result, it was confirmed that calcified deposits were observed and differentiated into osteoblasts (FIG. 3). Moreover, as a result of oil-red-O staining of UCMSC cultured for 32 days in a fat differentiation induction medium, it was found that cells containing oil droplets were recognized and differentiated into adipocytes (FIG. 3). On the other hand, tissue sections were prepared from UCMSC pellet-cultured for 21 days in a cartilage differentiation-inducing medium and stained with Alcian blue. As a result, positively stained tissues were observed, indicating that cartilage tissue was formed (Fig. 3). Furthermore, as a result of inducing neural differentiation of UCMSC for 12 days and fluorescent immunostaining, it was confirmed that neural progenitor cell marker positive cells were recognized and differentiated into neural progenitor cells (FIG. 3). As described above, UCMSC is a mesenchymal stem cell that can be differentiated into mesenchymal tissue, and was confirmed to be able to differentiate into a nerve beyond the germ layer.

3.HUVEC characteristic analysis <Method>
a. Flow cytometry Vascular endothelial cell surface markers are analyzed by flow cytometry. FACS Calibur (Becton Dickinson) was used for the analysis.

b. Fluorescent immunostaining Cultivate vascular endothelial cells using a dish coated with type I collagen. Cells are confirmed for vascular endothelial cell-specific markers by immunostaining with antibodies against CD31 and vWF.

<Result>
The collected cells were CD31 positive, CD34 positive, and CD45 negative. As a result of fluorescent immunostaining, they were stained CD31 positive and vWF positive. That is, it was confirmed that vascular endothelial cells were collected (FIG. 4).

4. Induction of UCMSC differentiation into osteoprogenitor cells <Method>
a. Alkaline phosphatase activity test
UCMSCs are seeded in 24-well adherent cell culture dishes at 3.1 × 10 ³ cells / cm ² . In order to induce differentiation of cells into osteoprogenitor cells in a short time, 50-300 ng / ml BMP-2 is added to the bone differentiation induction medium and cultured. During differentiation induction, medium is changed once every 3 days. Differentiation is induced for 1 week, and the alkaline phosphatase activity of the induced osteoprogenitor cells is analyzed using an alkaline phosphatase activity measurement kit (FAST p-Nitrophenyl phosphate tablet sets, Sigma). Absorbance values at 405 nm measured with a microplate reader should be divided by the absorbance value at 450 nm measured using a water-soluble tetrazolium salt WST-8 (Cell counting kit-8, Doujin Chemical). To correct the number of cells. As a control, human bone marrow mesenchymal stem cells (BMMSC, Lonza) are used.

b. Bone progenitor lineage marker analysis
UCMSC is added to bone differentiation-inducing medium at 100 ng / ml BMP-2, differentiation is induced for 1 to 2 weeks, RNA is extracted, and reverse transcriptase is used to prepare cDNA. Using this as a template, gene expression of osteoprogenitor cell lineage markers is analyzed by qualitative PCR using oligo primers that amplify ALP (Alkaline phosphatase), COLI (collagen type I), runx2, and osteocalcin (OSTEOCALCIN). The same analysis is performed using human bone marrow mesenchymal stem cells (BMMSC, Lonza) as a control.

<Result>
As a result of culturing UCMSC for 1 week after adding 50-100 ng / ml BMP-2 to a bone differentiation-inducing medium, a significant increase in alkaline phosphatase activity was observed (FIG. 5). In addition, as a result of adding 100 ng / ml BMP-2 to the bone differentiation-inducing medium and culturing, it was found that the bone precursor cell lineage marker of UCMSC was expressed (FIG. 5). Therefore, it was found that by adding BMP-2 to the bone differentiation-inducing medium, UCMSC can be induced to differentiate into osteoprogenitor cells in a short period of time.

5. UCMSC and HUVEC co-culture test <Method>
Mix 1 × 10 ⁴ each of UCMSC and HUVEC and inoculate in a 24-well adherent cell culture dish. 1 ml of MSCBM and EBM-2 mixed at a ratio of 1: 1 is injected into each well as a medium, and cultured in an incubator adjusted to 5% CO ₂ · 37 ° C. During the co-culture, the medium is changed once every 3 days. Co-culture for 1 week and analyze alkaline phosphatase activity using the same kit.

<Result>
It was found that alkaline phosphatase activity was increased by coculturing UCMSC and HUVEC for 1 week (FIG. 6). It is considered that HUVEC enhanced the ability of UCMSC to differentiate into bone. Thus, it was found that co-culture with HUVEC is effective in enhancing bone differentiation ability.

6. Induction of bone differentiation by application of hypoxic response of UCMSC <Method>
By using DFO (Deferoxamine), which inhibits VHL that ubiquitinates HIF-1, HIF-1 can be stabilized and artificially hypoxic. UCMSCs are seeded in 24-well adherent cell culture dishes at 3.1 × 10 ³ cells / cm ² . Pretreatment is performed by culturing in a basic medium supplemented with 10 to 50 μM DFO for 24 hours. After pretreatment with DFO, the medium is replaced with a bone differentiation-inducing medium to induce bone differentiation. During differentiation induction, medium is changed once every 3 days. Differentiation is induced for 1 week, and the alkaline phosphatase activity of the induced UCMSC is analyzed using the kit described above. As a negative control, a bone differentiation induction medium to which dexamethasone is not added is used. In addition, RNA of UCMSC that has been induced to differentiate for 1 week is extracted, and cDNA is prepared using reverse transcriptase. Using this as a template, HIF-1α, VEGF-A, BMP-2, VEGFR-1, VEGFR-2 and oligo primers that amplify the gene expression of hypoxia response factor and angiogenic factor by qualitative PCR To analyze.

<Result>
Hypoxia-treated UCMSC that had been pretreated with DFO and induced the expression of HIF-1 showed high alkaline phosphatase activity, suggesting that bone differentiation ability was enhanced (FIG. 7). Thus, it has been found that hypoxic treatment is effective in enhancing bone differentiation ability. In addition, gene expression of hypoxia response factor HIF-1α and angiogenic factor was observed by DFO pretreatment (FIG. 7).

7. Cell transplantation test 1 (Examination of bone formation ability using nude mouse ectopic ossification model)
<Method>
a. Preparation of cell transplant
1 × 10 ⁶ UCMSCs are suspended in a medium supplemented with 100 ng / ml BMP-2 in a bone differentiation induction medium. Mix 50 mg of hydroxyapatite granules (Calcitite, ZIMMER) and UCMSC cell suspension in a 15 ml polypropylene tube. After centrifuging for 5 minutes (800 rpm), incubate in an incubator adjusted to 5% CO ₂ · 37 ° C. The culture medium is changed once in 3 days with BMP-2 supplemented bone differentiation induction medium and cultured for 1 week. In the UCMSC and HUVEC mixed cell transplant, 0.5 × 10 ⁶ UCMSC and 0.5 × 10 ⁶ HUVEC mixed with MSCBM and EBM-2 at 1: 1 are suspended in a mixed medium. Mix 50 mg of hydroxyapatite granules and UCMSC / HUVEC cell suspension in a 15 ml polypropylene tube. After centrifuging for 5 minutes (800 rpm), incubate in an incubator adjusted to 5% CO ₂ · 37 ° C. The culture medium is changed once every 3 days and cultured for 1 week.

b. Transplantation into nude mouse ectopic ossification study model Nude mice (Balb / C, 5 weeks old, female, body weight 18-20 g) are anesthetized with Nembutal. An incision is made with a scalpel on the back, a pocket is formed under the back, a cell preparation is transplanted, and sutured with an absorbable suture to finish. After 6 weeks, the animals are sacrificed and the transplant is removed for histological evaluation.

<Result>
A hematoxylin-eosin stained image 6 weeks after transplantation is shown (FIG. 8). In the control (left), sufficient bone regeneration is not observed, but in the UCMSC transplant (middle) and UCMSC and HUVEC mixed cell transplant (right), bone regeneration is observed. In addition, an increase in bone tissue was observed in UCMSC and HUVEC mixed cell transplants. Thus, it was demonstrated that UCMSC is effective for bone regeneration. Moreover, it was shown that the combined use of HUVEC enhances the regeneration effect.

8. Cell transplantation test 2 (Investigation of therapeutic effect using nude mouse wound healing model)
a. Preparation of cell transplant
Transplant 1 × 10 ⁶ cells (UCMSC or 1: 1 mixture of UCMSC and HUVEC). In the cell transplant for subcutaneous injection, 0.7 × 10 ⁶ cells are precipitated by centrifugation, the supernatant is aspirated, and the pellet is suspended in 80 μl of PBS. The cell graft for wound coating is prepared by suspending 0.3 × 10 ⁶ cells pelleted in the same manner as described above with 20 μl type I collagen gel (Cellmatrix, Nitta Gelatin).

b. Transplantation into nude mouse wound healing model Nude mice (Balb / C, 8 weeks old, female, body weight 18-25 g) are anesthetized with Nembutal. A wound is made by excising the entire epidermis layer with a 6mm diameter biopsy punch in two places, one on each side of the back. A silicon plate (thickness 0.5 mm, outer diameter 14 mm, inner diameter 6 mm) is attached to the wound and fixed with an instantaneous adhesive, and the silicon plate and the surrounding skin are sutured with 4-0 nylon suture. Inject 20 μl of the cell implant for subcutaneous injection into a total of 4 locations using a Hamilton syringe. Further, the wound surface is covered with 20 μl of the wound cell coating. Finally, Tegaderm ^TM (3M) is attached for the purpose of protecting the wound. PBS is transplanted as a control.

c. Measurement of wound morphology Photograph the wound after transplantation with a camera daily and measure the wound area. The wound reduction ratio is calculated by (Wound area immediately after surgery−Area of daily wound area) / Wound area immediately after surgery × 100 (%).

<Result>
In the UCMSC transplant group and the UCMSC and HUVEC co-transplant group, an increase in the wound reduction rate after surgery was observed, and a difference from the control group was observed (FIG. 9). In the UCMSC and HUVEC co-transplanted group, an increase in wound reduction rate was observed. Thus, it was proved that UCMSC transplantation was effective for wound healing and the effect was enhanced by co-transplantation with HUVEC.

  In recent years, advancements in prenatal diagnostic techniques using fetal echo have made it possible to diagnose neonatal congenital diseases before birth. Cell collection from the umbilical cord can be performed non-invasively at birth, and autoserum purified from umbilical cord blood can also be used for cell culture. The composition of the present invention comprising an umbilical cord-derived mesenchymal stem cell as an active ingredient is a neonatal periventricular leukomalacia, which is a congenital neurological disease, a congenital heart disease that requires postpartum surgery or a congenital labial jaw It can be applied to the treatment of cleft palate. ADVANTAGE OF THE INVENTION According to this invention, the newborn regenerative medical system with respect to the congenital disease by the safe noninvasive autologous stem cell using an umbilical cord can be developed. The applicability of the present invention is not limited to these diseases. Examples of application cases of the composition of the present invention include bone diseases (periodontal disease, bone defects (bone augmentation for implants, jaw cleft, Including tumor excision), osteoporosis, fracture, ligament rupture, sports trauma, etc., cartilage disease (knee joint, temporomandibular joint, etc.), neurological disease (nerve regeneration: Alzheimer's disease, Parkinson's disease, etc.), skin disease (keloid, Application to scars), age-related diseases (anti-aging diseases such as wrinkles and bruises), and vascular disorders (such as revascularization) are also envisaged.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Claims (21)

  A composition for transplantation comprising umbilical cord-derived mesenchymal stem cells.   The composition for transplantation according to claim 1, wherein the umbilical cord-derived mesenchymal stem cells are derived from Wharton's jelly.   The composition for transplantation according to claim 1 or 2, which is used for treatment of neonatal periventricular leukomalacia, congenital heart disease or congenital cleft lip and palate.   The composition for transplantation according to claim 1 or 2, wherein the umbilical cord-derived mesenchymal stem cells are undifferentiated umbilical cord-derived mesenchymal stem cells not subjected to differentiation induction treatment after collection.   The transplant composition according to claim 4, wherein the umbilical cord-derived mesenchymal stem cells are CD73 positive, CD90 positive, CD105 positive, CD11b negative, CD34 negative, CD45 negative, and HLA-DR negative.   The composition for transplantation according to claim 4 or 5, which is used for regeneration of skin tissue or nerve tissue.   The composition for transplantation according to claim 1 or 2, wherein the umbilical cord-derived mesenchymal stem cells are differentiation-inducing umbilical cord-derived mesenchymal stem cells that have been induced to differentiate into a specific cell lineage.   The composition for transplantation according to claim 7, which is used for regeneration of bone tissue, cartilage tissue, nerve tissue, muscle tissue or vascular tissue.   The composition for transplantation according to claim 7, wherein the differentiation-inducing umbilical cord-derived mesenchymal stem cell has alkaline phosphatase activity, is positive for type I collagen, positive for RUNX2, and positive for osteocalcin, and is used for bone tissue regeneration. .   The composition for transplantation according to claim 7, wherein the differentiation-inducing umbilical cord-derived mesenchymal stem cells are cells in which alkaline phosphatase activity is enhanced by stimulation with bone morphogenetic protein 2 (BMP-2).   The composition for transplantation according to claim 7, wherein the differentiation-inducing umbilical cord-derived mesenchymal stem cells are cells in which alkaline phosphatase activity is enhanced by co-culture with umbilical vein endothelial cells.   The composition for transplantation according to claim 7, wherein the differentiation-inducing umbilical cord-derived mesenchymal stem cells are cells in which alkaline phosphatase activity is enhanced by culturing in a hypoxic environment.   The transplantable composition according to claim 12, wherein the culture in a hypoxic environment is a culture using a medium to which deferoxamine is added.   The composition for transplantation as described in any one of Claims 1-13 which combines a umbilical vein endothelial cell.   15. The composition for transplantation according to claim 14, comprising umbilical cord-derived mesenchymal stem cells and umbilical vein endothelial cells.   The composition for transplantation according to claim 14, which is a kit comprising a first component containing umbilical cord-derived mesenchymal stem cells and a second component containing umbilical vein endothelial cells.   The composition for transplantation according to claim 14, comprising umbilical cord-derived mesenchymal stem cells, wherein umbilical vein endothelial cells are transplanted together at the time of transplantation.   The transplant composition according to any one of claims 14 to 17, wherein the umbilical cord-derived mesenchymal cell donor and the umbilical vein endothelial cell donor are the same.   A method for repairing or regenerating a tissue, which comprises injecting, embedding, filling, or applying the transplant composition according to any one of claims 1 to 18 to a target site.   A method for preparing a cell with enhanced alkaline phosphatase activity, comprising a step of co-culturing umbilical cord-derived mesenchymal stem cells and umbilical vein endothelial cells.   A method for preparing a cell having enhanced alkaline phosphatase activity, comprising a step of culturing umbilical cord-derived mesenchymal stem cells in a hypoxic environment.

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