WO2003082305A1

WO2003082305A1 - Drug containing human placenta-origin mesenchymal cells and process for producing vegf using the cells

Info

Publication number: WO2003082305A1
Application number: PCT/JP2003/004006
Authority: WO
Inventors: Naohide Yamashita; Takashi Nakaoka; Toshihide Nishishita
Original assignee: Naohide Yamashita; Takashi Nakaoka; Toshihide Nishishita
Priority date: 2002-04-03
Filing date: 2003-03-28
Publication date: 2003-10-09
Also published as: JPWO2003082305A1; JP4554940B2; AU2003220966A1

Abstract

A drug containing human placenta-origin mesenchymal cells; and a process for culturing human placenta-origin mesenchymal cells characterized in that the cells are cultured in a medium containing human serum albumin and cell growth factor but being free from any protein components originating in nonhuman animals.

Description

Medicament containing mesenchymal cells derived from human placenta and VE using the cells

Manufacturing method of GF

TECHNICAL FIELD ffl The present invention relates to use of human placenta-derived mesenchymal cells, in particular, a pharmaceutical composition for treatment of ischemic disease containing the cells, a method for producing vascular cell growth factor using the cells, the cells It is related with the method of culture | cultivating. Technical background Ischemic diseases, such as myocardial infarction and cerebral infarction, occupy a high level of human death, and active research is being conducted on both surgical and pharmaceutical prevention and treatment methods. A typical example of the surgical method is a vascular bypass procedure, such as percutaneous coronary angioplasty (PTCA) or coronary bypass (CABG). In addition to thrombolytic agents such as t-PA and thrombin, pharmacological methods include VEGF (V ascu 1 ar E), a factor that promotes angiogenesis in vivo. ndothelial G rowth F actor; vascular endothelial growth factor) and FGF (Fibroblast Gr oeth F actor: Basic fibroblast growth factor) is being tried.

In recent years, as a method of relieving the ischemic state by creating a new blood vessel at the ischemic site and reproducing the blood flow, a specific cell is used instead of the above growth factor. Angiogenic treatment by cell transplantation is attracting attention. Living organisms exposed to prolonged ischemia have the ability to create new blood vessels with their own power to secure blood flow. By introducing it, it is intended to promote angiogenesis more effectively and to be used for the treatment of ischemic diseases.

Cells that are expected to be applied to this method include vascular endothelial cells that are known to make a significant contribution to angiogenesis, or bone marrow stem cells and mononuclear cells that have the ability to differentiate into these cells. is there. Vascular endothelial cells are cells that form a monolayer covering the lumen of blood vessels, and are closely related to angiogenesis during wound healing and tumor growth, and their presence is regarded as important. To date, vascular endothelial cell proliferation has been implicated in diseases with angiogenesis such as tumor growth, progression of retinopathy or rheumatoid arthritis, or the expansion of psoriasis. Therefore, focusing on the suppression of the proliferation of vascular endothelial cells Research has been underway.

On the other hand, treatment for diseases that can be expected to recover from disability by promoting angiogenesis is also being studied. In particular, cell endothelium is attracting attention as an advantageous cell because it has a function of secreting a protein that promotes angiogenesis in addition to its proliferative ability. So far, in animal models (myocardial infarction model, angina pectoris model, obstructive arteriosclerosis model) experiments, angiogenesis was induced by adding vascular endothelial cells to the model animal, and decreased due to ischemia. In addition, it has been reported that the function of organs is improved, and attempts have been made to induce angiogenesis by transplanting vascular endothelial progenitor cells present in the peripheral blood of adults into the body. Vascular endothelial progenitor cells are isolated as CD 34-positive cells, but have been confirmed to differentiate into endothelial cells in vivo. In particular, it has been reported that these CD 3 4 positive cells are present in umbilical cord blood at a density approximately 10 times higher than that in adult peripheral blood. Attempts have also been made to induce neovascularization by transplanting endothelial progenitor cells into the body. When vascular endothelial progenitor cells obtained on day 7 of umbilical cord blood mononuclear cell culture were transplanted to the unilateral droop of lower limb ischemia after isolation, vascular endothelial progenitor cells A significant increase in blood flow and improvement in capillary density was reported in the transplanted group.

The present invention provides new cells effective for angiogenesis treatment, which are different from previously reported vascular endothelial cells or vascular endothelial progenitor cells.

Brief Description of Drawings

Fig. 1 shows the growth of mesenchymal cells in the culture method of the present invention. Fig. 2 shows the results of measuring the activity of VEGF produced in the medium. Fig. 3 shows the intramuscular muscles of a nude mouse. It is a microscope picture showing the appearance of angiogenesis. Fig. 4 shows the result of fluorescent immunostaining using anti-VEGF antibodies between mesenchymal cells and comparative cells.

Figure 5 shows the blood flow improvement effect observed after transplanting mesenchymal cells into a mouse hindlimb ischemia model using a laser Doppler blood flow meter.

Fig. 6 shows the results of confirming VEGF production in the transplanted cells and the amount of VEGF mRNA in the transplanted tissue using the real-time RT-PCR method. Disclosure of the invention

The placenta is a place for nutrient exchange between the fetus and the mother, and there are abundant blood vessels. The present inventor focused on a group of stromal cells (cells other than vascular endothelial cells) present in the placenta that are usually discarded together with the umbilical cord at the time of birth of a human, and mesenchymal cells contained therein The inventors have found that an angiogenic action can be promoted in a living body by transplanting and administering to the living body, and the present invention has been completed. That is, the present invention is a medicament for treating ischemic disease, comprising mesenchymal cells derived from human placenta. The present invention also relates to a method for culturing mesenchymal cells, which is advantageous in preparing the pharmaceutical, and a method for producing VEGF using the mesenchymal cells.

BEST MODE FOR CARRYING OUT THE INVENTION

Unlike mesenchymal cells such as vascular endothelial cells and vascular endothelial progenitor cells, which are histologically constituting connective tissue, mesenchymal cells are generally pluripotent cells having multiple differentiation potentials. In particular, mesenchymal stem cells are known to be bone, cartilage, fat, heart, nerve, liver cells, etc., differentiated fibroblasts, hair papilla cells, adipocytes, pulp cells, etc. Also belong to mesenchymal cells.

There are no reports that some mesenchymal cells are involved in various diseases. Has been. For example, it has been reported that mesenchymal cells present in the synovium are involved in joint destruction such as rheumatoid arthritis and collagen disease. In this case, suppression of the activity of synovial mesenchymal cells has been attempted in the treatment of joint disorders.

However, human placenta-derived mesenchymal cells have the effect of promoting angiogenesis in vivo, and no attempt has been made to positively use this for the treatment of imaginary disease. .

The mesenchymal cells derived from human placenta used in the present invention can be treated by ordinary cell separation using an enzyme such as trypsin after the human placenta is cut into sections of an appropriate size. Therefore, it can be isolated easily and does not require any special operation. The human placenta itself is generally treated as a waste product during childbirth, and is a separation material that can be obtained on a daily basis in the medical field.

Isolated mesenchymal cells can be grown in an appropriate medium. Cultivation is generally 33-39 ° C, preferably 37 ° C, and fetal bovine serum, preferably inactivated fetal bovine serum (by heat treatment, A basal medium containing 3 to 10% (preferably 10%) of complement-inactivated fetal bovine serum (for example, α-MEM medium) may be used. The ventilation uses air containing 5% C 0 ₂ , Cultivation can be carried out at a humidity of 80 to: L 20% (preferably 100%).

Mesenchymal cells can be preserved by a conventionally known method. For example, 10 ⁵ to 10 ⁸ cells / ml in a nutrient medium containing 10% glycerin or 10% dimethyl sulfoxide and 10% serum, preferably 10 ⁶ ~ 10 ⁷ cells / ml, more preferably 5 x 10 ⁶ cells / m 1, stored frozen at _80 ° C or liquid nitrogen at a cell concentration of 5 x 10 ⁶ cells / m 1 You can. The stored cell line is rapidly lysed (for example, immersed in a 37 ° C water bath), and 10 times the same medium is added, stirred, and centrifuged to recover the recovered cells. By adding it to the ground, it can grow again.

In general, when transplanting cells themselves into a living body, it is necessary to pay attention to the agreement of the HLA activity between the transplanted cells and the living body to be transplanted so as not to induce an undesirable immune response. Therefore, also in the present invention, it is preferable to select a mesenchymal cell for transplantation that matches the HLA type of the patient undergoing transplantation and administer it as the medicament of the present invention. In the case of the medicine containing human placenta-derived mesenchymal cells according to the present invention, leukocyte treatment or the like using umbilical cord blood is performed. HLA haplotype of umbilical cord blood accumulated for so-called umbilical cord blood transplantation Since this represents the haplotype of mesenchymal cells as it is, there is an advantage that the umbilical cord blood HLA event information can be used as it is.

(Measuring method of mesenchymal cells)

Mesenchymal cells isolated from the placenta can be used immediately after washing with an appropriate buffer, or they can be used immediately after suspending in a buffer. It is preferable to grow the cultured cells. Human placenta-derived mesenchymal cells can be grown in commonly used nutrient media. However, considering that the cultured cells are finally administered to humans, mesenchymal cells can be cultured under conditions that do not contain foreign substances that cause immune responses to humans. I prefer to do it. The present invention provides a culture method for solving this problem.

The culture method of the present invention is characterized by culturing in a medium not containing a protein component derived from a non-human animal to which human serum albumin and a cell growth factor are added. This is a method for culturing cell lines.

In general, MEM medium, RPMI 16 40, RITC 80 — 7 MCDB series, H am F — 1 2. DME 1: 1 mixed medium, etc. In this case, since the growth of the isolated cells does not progress only in the basal medium in which amino acids are added to amino acids, the serum from non-human animals such as pups of fetuses and pups is not available. Add to basal medium. However, in this culture method, although cell growth itself is performed well, administration of the cultured cells into the human body is preferable for a living body to which a mixed non-human protein is injected. Often elicits a rare immune response. In addition, the effects of various physiologically active substances contained in serum are complex and diverse, and the serum lot difference is severe, so that cultured cells with stable properties or pharmaceuticals containing them can be used. There are many disadvantages to the supply of

The present invention includes a substance derived from a non-human animal by adding human serum albumin and a cell growth factor to the basal medium as described above that does not contain an element derived from an animal other than human. It is intended to culture mesenchymal cells in a non-environmental environment. Growth factors used in the present invention include platelet-derived growth factor (PDGF, Platelet-Derived Growth F actor), fibroblast growth factor (b FGF basic Fibroblast G rowth F actor), epithelial cell growth factor. (EGF, E piderma 1 G rowth F actor) and insulin One or more selected from the group. These growth factors may be commercially available, for example, as research reagents from R & D or Sigma, and are produced by separation and purification from living organisms or by gene recombination techniques. It may be a thing. The added amount is from 0.01 to: LOOO ng Zml, preferably 0.1 to:! OO ng / ml, most preferably 1 ~! O ng Zm l.

Human serum can be prepared from the donated blood by centrifugation. The amount of human serum added is about 1 to 20% of the culture medium, preferably 2 to; L 5%, and most preferably 5 to 10%. When using formulated human albumin: 0.01 ~: L 0%, preferably 0.05 ~; L%, most preferably 0.5:! ~ 0.5 %.

Furthermore, by adding an antioxidant, particularly an antioxidant selected from the group consisting of 2-mercaptoethanol, ascorbic acid and vitamin E, to the medium, Proliferation can be further enhanced. The amount of antioxidant added in the case of 2-Merkabutanol is from 0.01 to 10:00 〃 1 ^, preferably 0. ~ 50 M, most preferably 1 ~ 1 1 ^ Μ. For ascorbile acid or bibutamin 量, an amount equivalent to the above 2—merkabutanol Can be used.

The medium and the culture method according to the present invention can be used in both primary culture and subculture of fibroblasts, and good results are obtained.

(Medicine containing mesenchymal cells)

The medicament containing mesenchymal cells of the present invention uses mesenchymal cells isolated from human placenta or mesenchymal cells cultured by the method described above, and a suitable pharmaceutical carrier or excipient for this. It can be prepared by adding. Since a neovascularization is observed at the injection site by injecting the medicinal cell-containing drug of the present invention into the thigh of a nude mouse, the drug is locally injected into the ischemic site. As a result, angiogenesis is promoted at the site, a blood flow path is acquired, and blood supply to the ischemic site can be resumed. Therefore, the angiogenesis-promoting agent of the present invention is useful as a therapeutic agent for ischemic diseases such as myocardial infarction, angina pectoris, cerebral infarction, vascular dementia, obstructive arteriosclerosis, foot gangrene and sores. . It is preferable to administer intravascularly, intramuscularly or locally, and also intramyocardial, intrapericardial, subdural, subarachnoid, intracerebral Particularly preferred is local administration to the ischemic region such as administration or intralimb skeletal muscle administration. The preferred pharmaceutical form is a drip or injection. These infusions or injections are preferably prepared by mixing mesenchymal cells in physiological saline, buffer solution, phosphate buffered saline (PBS) or the like. In addition, stabilizers, preservatives, excipients, etc. may be added depending on the purpose.

In addition, a cell growth factor that promotes angiogenesis may be added to the medicament of the present invention. These cell growth factors include acidic and basic fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and transforming factor. Growth factor (TFG and /?), Platelet-derived endothelial growth factor (PDEGF), platelet-derived growth factor (PDGF), tumor necrosis factor (TNF), hepatocyte growth factor (HGF), insulin-like growth Factor (IGF), erythropoietin, colony stimulating factor (CSF), macrophage CSF, granulocyte Z macrophage CSF and nitric oxide synthase, angiopoietin Angios Yuchin, etc.

The dose of the medicament of the present invention varies depending on the medical condition, age, weight, etc. of the patient receiving the transplantation, but is applied in the range of, for example, 10 ⁵ to 10 ⁸ cells per administration. be able to. In addition, the mesenchyme of the present invention Since the cells are cells derived from living bodies, they can be used as pharmaceuticals without worrying about the toxicity of the cells.

(V E G F manufacturing method)

The present invention relates to a method for producing VEGF using human placenta-derived mesenchymal cells.

As mentioned earlier, it was reported that vascular endothelial cells themselves express and secrete vascular endothelial cell growth factors, but mesogenic cells that are different from vascular cells express VEGF. It was quite surprising that parentheses were extracted outside the cell.

In the production of VEGF using the mesenchymal cells of the present invention, it is only necessary to culture the cells in a medium in which the isolated mesenchymal cells can proliferate, and this culture can secrete VEGF into the medium. The VEGF produced and secreted by mesenchymal cells has biological activity, and angiogenesis was caused in the mouse even in an in vivo experiment of a mouse.

As a medium for producing VEGF, a medium such as MEM medium, RPMI 16 40, RITC 80 — 7, MCDB series, H am F — 1 2 · DME 1: 1 mixed medium, etc. 5-10% mammalian serum (eg, A medium supplemented with fetal calf serum, calf serum, human serum, or horse serum) is preferred, but it contains a cell growth factor that does not contain non-human-derived components as described above. Also good.

The cells may be cultured from the beginning in a serum-free medium, or cultured to a subconfluent in a medium containing serum from non-human animals, then the medium is removed and phosphate buffered saline solution is dissolved. The cell layer may be washed with a solution and replaced with a serum-free medium for cultivation. The other culture conditions are not special. Atmospheric pressure, 37 ± 0.5 ° C, 5% carbon dioxide as gas phase, 10% to 20% oxygen, 85% to 75% nitrogen If it is%.

From the culture supernatant obtained by culturing mesenchymal cells in this manner, VEGF can be recovered according to a general method. In addition, an appropriate method can be selected appropriately from methods usually used for protein purification. That is, salting-out method, ultrafiltration method, isoelectric point precipitation method, gel filtration method, electrophoresis method, ion exchange chromatograph, hydrophobic chromatograph and antibody chromatograph Various affiliation chromatograms such as graphs, chromatofocusing methods, adsorption chromatographs and reversed phase chromatographs Appropriate method is selected from methods that can be used normally. If necessary, purification can be performed in an appropriate order using an HPLC system or the like.

(Recombinant mesenchymal cells)

The mesenchymal cells used in the present invention can be transformed using an appropriate vector, particularly a virus vector. In that case, protein factors effective for the improvement of hemorrhagic disease, acidic and basic fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), transfusion -Ming growth factor

(TFG and 5), platelet-derived endothelial growth factor (PDEGF), platelet-derived growth factor (PDGF), tumor necrosis factor (TNF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF) , Erythropoietin, colony-stimulating factor (CSF), macrophage CSF, granulocyte nomacrophage CSF and nitric oxide synthase, angiopoietin, angios yutin, etc. Can be incorporated into a vector and the gene can be expressed in mesenchymal cells. In vivo gene therapy and ex vivo gene therapy are methods for treating diseases by administering cells into which this gene has been introduced to patients. The latter is a significant burden on the patient for surgical procedures and the risk of infection due to extracorporeal manipulation. In vivo gene therapy is preferred because of its steep nature.

The main viral vectors in clinical application are the retrovirus vector and the adenovirus vector, and recently attracted attention is the adeno-associated virus vector.

In the retrovirus vector, since the transgene is integrated into the chromosome, stable gene expression can be expected for a long period of time. In addition, it is advantageous in that a large number of various types of virus vectors can be easily produced using the packaging cells.

Adenovirus vectors have a 36 kb linear double-stranded DNA as a genome, are physicochemically stable, and can be easily concentrated by ultracentrifugation, resulting in extremely high efficiency. The gene can be introduced with. However, since the transgene is not integrated into the chromosome, it is difficult to retain the recombinant gene for a long period of time, and long-term gene expression cannot be expected. The denovirus vector must be administered repeatedly, and inactivation by neutralizing antibodies is a major problem.

The adeno-associated virus vector is a virus that has a 4.7 kb single-stranded DNA and is not a replication virus that itself does not have the ability to replicate. Pesu A superinfection of virus is necessary. In other words, the adeno-associated virus itself has no pathogenicity or cytotoxicity, so it is the safest virus vector, and since the recombinant gene is integrated into the host chromosome, Gene expression is possible.

Methods for introducing a desired DNA into these vectors are known (for example, J. Sambrook et al., Mo 1 ecular C 1 oning, a Laboratory 2 nded., Old Sprung Harbor Laboratory , New York, 1 989, see). That is, DNA and vector can be digested with appropriate restriction enzymes and the resulting fragments can be ligated using DNA ligase. In addition, a method for transforming mesenchymal cells using these virus vectors can be performed by a method well known by those skilled in the art, for example, the method of Saito et al.

EXAMPLES Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

Example

Example 1 Isolation of human placenta-derived mesenchymal cells After collecting the placenta produced at full term on ice, placenta sections from which the tissue was collected with scissors from the maternal side of the placenta were transferred to a beaker, and 0.5% trypsin, 5.3 mM EDTA l Add 1 L of DMEM medium with OO ml and isolate cells at room temperature. Mononuclear cells were separated using Ficoll — Hypaque (P harmacia Biotech AB). This which was suspended in DMEM solution, and cultured 5-7 days at 3 7 ° C, 5% C 0 2 under - the mononuclear 1 0% FBS in earthenware pots by that Do and 1 XI 0 ⁶ cells / m 1 As a result, desired cells can be prepared. The cultured cells thus obtained can be used for the following operations. Hereinafter, the four lines of cells actually separated by the present inventors will be referred to as PL 5, PL 24, PL 26 and PL 37, and the following examples will be described in detail. Example 2 Culture method of mesenchymal cells using serum-free medium

The mesenchymal cells PL 37 that had been collagen-coated in a 10 cm dish and cultured in the same manner as in Example 1 were detached using 0.25% trypsin. After stopping the trypsin reaction by adding 10 ml of PBS containing 5% FCS, the cells were washed twice with MCDB 10 4 medium. MCDB 10 4 medium contains a final concentration of 2 mM glutamine, 1 of 2 —merkabut ethanol, 0.0 1 mg / ml insulin and 5 mg / ml human serum albumin were added. After counting the number of cells, a cell solution of 1 XI 0 ⁴ cells / 500 1 was prepared.

PDGF — AB, b FGF, EGF and FCS, which are growth factors, are placed in a 96-well plate at twice the final concentration shown in Table 1/5 0 1 in advance. In addition, the cells were cultured at 37 ° C for 72 hours, and the state of cell proliferation was examined using XTT assay (Cell Proliferation Kit II, Roche Diagnostics). As a control, a medium containing no cell growth factor and a medium containing 10% FCS were prepared. The results are shown in Figure 1

PL 37 cells can grow in serum-free MCDB 104 medium supplemented with PDGF-AB, bFGF, EGF, and the growth is promoted more than in MCDB medium supplemented with 10% FCS. It was. In addition, by adding 2-mercaptoethanol and insulin, the growth promoting effect became more prominent. Also, Regarding the growth of PL 37 cells in serum-free medium, the optimal concentration was observed in the growth factor concentration, and the results showed that the cell growth slightly slowed before and after the optimal concentration. It was also found that the growth factor action was lower than that of 10% FCS in the absence of albumin.

Example 3 Production of angiogenic factor (V E G F) from human placenta-derived cells

2 to 5 x 10 ⁶ cells of PL 5 and PL 24, which are mesenchymal cells of the present invention, in 37 ml of DMEM + 10% FCS, respectively, at 37 ° C and 5% C 0 2 The cells were cultured under passage 10 or below. PL 26 was cultured under 5 passages under the same conditions.

The culture supernatant was collected over time, and VEGF in the medium was measured with a Quantikine VEGF measurement kit (R & D). In addition, according to the method of Conn et al. (Pro. Natl. A cad. Sci. USA, 87, pp. 1 3 2 3, 1990) The activity was measured by a symidine uptake experiment. Furthermore, 5-week-old male Nu de mouse (balb / c, nu / nu ) were approximately 5 XI 0 ⁶ or injected PL 2 6 cells thigh muscle in. 1 After 4 days, sacrifice and fix the femoral muscle with paraformaldehyde, prepare the section, and then stain with HE Observed.

VEGF was detected in the supernatant of PL5 cells cultured for 2 days, and the production was increased particularly in the presence of FCS. As with VEGF, hepatocyte growth factor (HGF, hepatocytegrofahtofactor) having angiogenic activity was not detected. From the results of thymidine uptake experiments using the culture supernatant (Fig. 2), VEGF produced by these mesenchymal cells had the activity of stimulating the proliferation of HUVEC cells.

In addition, the number of cells when cultured in DMEM serum-free medium in 10 cm dish for 24 hours was counted, and V E G F in the supernatant was measured. The human placenta-derived mesenchymal cells in the present invention, P L 26 and P L 5 cells, produced a higher concentration of V E G F than He La cells. In addition, angiogenesis was observed under the microscopic observation in the muscle of the thigh of the nude mouse injected with PL 26 cells (Fig. 3), and VEGF produced by mesenchymal cells was also active in vivo. Became clear.

Example 4 Fluorescent immunostaining

Anti-VEGF antibody (Santa Cruz Biotechnology Inc. Santa Cruz, CA) is used as the primary antibody. And fluorescent immunostaining was performed. HeLa cells were used as a positive control, and normal human fibroblasts were used as a negative control. 1 Place a 12 mm l-type collagen coat cover glass in the 2-well plate and inoculate the cells. After incubation in a C 0 ₂ incubator for 2 4 — 48 hours, the cover glass was washed twice with PBS and fixed with 4% paraformaldehyde for 1 minute at room temperature. Thereafter, pretreatment with 0.3% hydrogen peroxide for 5 minutes at room temperature was performed to inactivate endogenous peroxidase activity. In subsequent steps, 0.1% Tween 20-PBS (PBST) was used for washing, and PBST with 2% BSA was used for antibody dilution. The primary antibody was allowed to react in a humidified box for 2 hours at room temperature, then washed 3 times with PBST, reacted with piotylated anti-rabbit IgG antibody for 30 minutes at room temperature, washed 3 times with PBST, and then FITC-labeled. Treftoavidin was allowed to react for 15 minutes at room temperature, protected from light. The sample was gently washed with PBST without being shielded from light, sealed using VECTASH IELDM Mounting Medium with DAPIV Laboratories, Inc., Burlingame, USA), and observed under a fluorescence microscope. Figure 4 shows the result.

In the figure, a and b are HeLa cells, c and d are human fibroblasts, e and f is a human placental cell, the left side is a DAPI corner observation, and the right side is a FITC colored cell with the same visual field. The mesenchymal cells of the present invention were also stained with VEGF antibody in the same manner as the positive HeLa cells. In this state, VEGF was not confirmed in fibroblasts, which are negative cells.

Example 4 Mouse hindlimb ischemia model and cell transplantation

A unilateral hindlimb ischemia model was prepared using N O D Z s cid mice (Claire Japan).

Xylazine 15 mg / kg intramuscularly and chemoprotein g Omg Z kg Intraperitoneally administered mice were ligated to the left femoral artery and vein of the mouse with 40 silk threads. Blood vessel ligation was performed under a stereomicroscope, and nerves were preserved without ligation. Seven days after the operation, the whole body was irradiated with 3 Gy and placental cells were transplanted. PL 2 6 and PL 3 7 were used as transplanted cells. Cells are suspended in a 0.5% solution of a type I collagen solution, Ce 11 1 gen (K oken, Japan), at a concentration of ⁶ lxl O / ml. One inoculated intraductally and subcutaneously. After creating an ischemic condition, the control mouth was inoculated with 0.5% collagen solution instead of cells. 1) Measurement of blood flow in the hind limb with a laser-doubler blood flow meter Improving blood flow with transplanted cells by measuring hindlimb blood flow in a prone mouse with a laser-dobler-flow meter (Moor LDI v3.08; Moo Instruments, UK) The operation was confirmed. Figure 5 shows the typical results.

The upper part of the figure shows hindlimb blood flow measurement results using a laser Doppler blood flow meter of a control mouse without cell transplantation, but compared to the left limb without ischemia. The ischemic right limb does not improve ischemia during the 6-day observation period. However, in the human placental cell-transplanted mice shown in the lower row, blood flow is restored from the second day after transplantation, and the improvement is remarkable on the sixth day. Thus, human placental cells also exhibited angiogenic effects in vivo.

2) Real time R T— P C R

The amount of VEGGF production in the transplanted tissue was confirmed by using the real-time RT-PCR method to determine how long the transplanted cells had been producing VEGFF.

The primer was created on the 3 'untranslated region based on the cDNA sequence of human VEGF (Gen Bank: AF 0 2 2 3 7 5). The sequences are hVLl: GGTCCCTCTTGGAATTGGAT, and hVRl: TGTATGTGGGTGGGTGT It is GTC and the PCR fragment length is 1 15 bp. Total RNA was extracted from cultured PL cells using Tri-zol (Invitrogen) according to the instructions. RT 1 〃 1 Oh Li GORE _{_{(d T) 12. 18 (}} 0. 5 〃 GZ〃 l), 2 〃 1 l O mM d NTP, 5 〃 g of DEPC water was added to total RNA total amount 1 2 〃 1 Then, it was incubated at 65 ° C for 5 minutes and placed on ice. In addition, 4 U 1 5 xc DNA synthesis buffer (Invitrogen), 0.1 MDTT (Invitrogen), 1 1 RNA seinhibitor (40 U / u 1) (Invitrogen), 1 〃 1 DEPC water, T hermo Script RNase H—Reverse Transcriptase, Invitrogen) was added to a total volume of 20 〃 1 and reacted at 50 ° C for 1 hour, then 85 ° C Incubated for 5 minutes. The reaction solution composition and reaction time used for PCR are as follows.

Reaction solution composition: 10 1 1 2 x SYBRG reen PCR mstermix (Qiagen), 1 〃 1 10 0] \ [h VL 1, 1 1 10 M h VR l, 1 1 template ( RT reaction solution above), 7 〃 1 water. Reaction conditions: 95 ° C, 15 minutes, 94 ° C, 30 seconds, 56 ° C, 30 seconds, 72 ° C, 30 seconds: 34 cycles, 72 ° C, 7 minutes

A portion was electrophoresed on a 2% agarose gel and stained with ethimubu mouth-mide.

The change over time in hVEGFmRNA was examined as follows. PL 37 cells were injected intramuscularly into SCID mice (Japan Clear) 3 hours later, on day 1, day 3 and day 7, the adductor muscle, the site of inoculation, was removed after sacrifice. , — Stored at 80 ° C. Extraction of total RNA from the tissue was performed using homogenized Tri 1 Ο 1 and RT was performed as described above. For PCR, real-time quantitative PCR was performed using iCyc1er (B1 0-RAD). The composition of the reaction solution and the reaction time are as follows.

Reaction solution composition: 2 5 l 2 x SYBRG reen PCR mastermix (Qiagen ¾ :), 2.5 j 1 10 j j M h VL 1, 2.5 ju I 10 0 ^ M h VR l, 1 1 tempiate ( RT reaction solution above), 19 〃 1 water.

Reaction conditions: 9 5 ° C for 15 minutes, 94 ° C for 30 seconds, 56 ° C for 30 seconds, C 2 ° C for 30 seconds for 45 cycles, 95 ° C for 3 minutes

5 5 ° ○ Increase the temperature by 0.5 ° C in steps of 10 seconds each. 8 0 cycles.

As a standard, a dilution series was prepared using an RT reaction solution of P L 37 cells. Figure 6 shows the result.

The amount of human VEGF mRNA was defined as 1 on day 0 (3 hours after transplantation), 0.20 on day 1, 0.22 on day 3, and 0.06 on day 7. The control not to be performed was 0.

From this result, it is considered that the transplanted cells have the ability to produce human VEGF for at least 5 days.

Claims

The scope of the claims

1. A medicament for treating ischemic diseases, comprising mesenchymal cells derived from human placenta.

2. The medicament according to claim 1, wherein the ischemic disease is myocardial infarction, angina pectoris, cerebral infarction, vascular dementia, obstructive arteriosclerosis, foot gangrene or wound.

3. The medicine according to claim 1 or 2, comprising a pharmaceutically acceptable carrier or diluent capable of being injected or instilled.

4. The medicine according to any one of claims 1 to 3, which is in a unit dosage form comprising ⁸ XI 0 ⁴ to 1 XI 0 ⁸ mesenchymal cells.

5. A method for culturing human placenta-derived mesenchymal cells characterized by culturing in a medium containing no serum component derived from non-human animals to which human serum and cell growth factor are added.

6. The culture method according to claim 5, wherein the human serum is human albumin.

7. The cell growth factor is at least one selected from the group consisting of platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and insulin. The culture method according to claim 5 or 6.

8. The culture method according to any one of claims 5 to 7, wherein an antioxidant is further added.

9. The culture method according to claim 8, wherein the antioxidant is selected from the group consisting of 2—merkabutanol ethanol, ascorbic acid and bibinmin E.

10. A method for producing VEGF, comprising culturing mesenchymal cells derived from human placenta and collecting the culture supernatant.

11. The method for producing VEGF as described in claim 10, wherein the culture is carried out in a medium containing serum albumin and cell growth factor and not containing a protein component derived from non-human animals.

1 2. The production method according to claim 10 or 11, wherein an antioxidant is further added.

1 3. The production method according to claim 12, wherein the antioxidant is selected from the group consisting of 2—mercaptoethanol, ascorbic acid, and vitamin E.