KR20070070445A - Method of preparing cell for heart tissue regeneration - Google Patents

Abstract

A method for preparing a cell for heart tissue regeneration is provided to optimize engraftment and survival rate of the cell in the heart muscle, and differentiate the cell into the heart muscle, so that myocardial infarction is prevented and treated. The method for preparing the cells for heart tissue regeneration comprises the steps of: (a) supplying immortalization-inhibited bone marrow stem cells; (b) subculturing the bone marrow stem cells while the cells maintain characteristics of the bone marrow stem cell; (c) culturing the bone marrow stem cells in a cardiac specific medium which induces commitment of the bone marrow stem cell into a cardiomyocyte while the cell maintains characteristics of bone marrow mesenchymal stem cell until 80-90% of the bone marrow stem cells are induced into the cardiomyocytes; and (d) collecting the cells of step(c) when the cultured cells show a marker specific to the bone marrow mesenchymal stem cell.

Description

Cell production method for myocardial tissue regeneration {METHOD OF PREPARING CELL FOR HEART TISSUE REGENERATION}

1 is a bone marrow stem cell surface protein analysis of Passage 0,

Figure 2 is an uncommitted bone marrow stem cell surface protein analysis of Passage 2,

Figure 3 is a picture of the analysis of committed bone marrow stem cell surface protein treated with growth factors in the bone marrow stem cells of Passage 2 6-7 days,

Figure 4 is a mixed culture of uncommitted bone marrow stem cells (A-D) and committed bone marrow stem cells (E-H) with myocardial blast cells for 2 days, the cardiomyocyte specific markers Connexin-43 (green) and cardiac Troponin I ( Fluorescence micrograph (400x), which measured differentiation of bone marrow stem cells by staining red),

5 is a laser fluorescence micrograph (400 times) of the analysis of the function of the gap junction formed by Connexin-43 expressed in the mixed culture with myocardial blast cells, (A) ~ (C) is mixed with uncommitted bone marrow stem cells Cultured photographs, (D) to (E) are mixed cultured with committed bone marrow stem cells, (G) to (H) is treated with heptanol in each group to inhibit the gap junction by Connexin-43 It is a photograph observing the movement of Calcein AM stain.

Figure 6 is a photograph showing the expression of the cardiomyocyte specific markers Nkx2.5, GATA-4, and MEF2 protein through Western blot experiment for uncommitted bone marrow stem cells and committed bone marrow stem cells, the fifth line of the rat Protein extracted from the heart and used as a positive control.

7 is a picture showing the differentiation into myocardial blast cells through the immunostaining method after 8 weeks after injection of committed bone marrow stem cells into the heart of myocardial infarction rat, the red one shows the committed bone marrow stem injected by staining with DiI stain solution. Cells, green indicates cardiomyocyte specific markers Connexin-43 (A, B; 200-fold), cardiac Troponin I (C-D; 200-fold), and MF-20 (E; 400-fold); Graphs showing the frequency of rats expressing each marker protein are gray for uncommitted and black for committed bone marrow stem cells.

8 is an 8-week echocardiogram of uncommitted bone marrow stem cells (A) and committed bone marrow stem cells (B) implanted in the heart of myocardial infarction rats, showing the end- and end-stem axial distances of the rats. , (C) is a left ventricular ejection fraction (LVEF), and (D) is a graph comparing left ventricular fractional shortening (LVFS).

9 is a result of measuring the degree of arrhythmia induced by injecting an arrhythmia-inducing substance in the rat injected with bone marrow stem cells, (A) shows the incidence of ventricular tachycardia, (B) shows early ventricular contraction.

The present invention relates to a method for producing a cell for transplantation into mammalian myocardial tissue, and more specifically, to a biological bone marrow stem cells of mammals, including humans, by treatment of biological growth factors directly committed to the cardiac lineage (cardiac lineage) In order to maintain the properties of the stem cells to ensure the best engraftment and survival rate in the damaged myocardium, the cells were treated in vitro for a certain period of time, and the induced bone marrow stem cells were transplanted into the heart of a mammal with myocardial infarction. The present invention relates to a method for evaluating differentiation and improvement of cardiac function from cardiac tissue to cardiomyocytes which can communicate with peripheral tissues.

Mesenchymal stem cells obtained from adult bone marrow have the ability to differentiate into various cells, including adipocytes, bone cells, chondrocytes, liver cells, and cardiomyocytes. These differentiation abilities can make bone marrow stem cells a good candidate for use in cell transplantation therapies.

Myocardial infarction, meanwhile, is known as one of the leading causes of death worldwide. Because myocardial blasts cannot proliferate in the adult heart, cardiomyocyte death due to myocardial infarction leaves a scar in the place of healthy cells.

Thus, the fundamental treatment for treating degraded heart function is to replace dead heart tissue with healthy cardiomyocytes.

Recent studies have shown that stem cells such as embryonic stem cells or adult stem cells have incredible flexibility after transplantation into the heart. When stem cells are transplanted into the myocardium, they have the ability to be transdifferentiated into myoblasts, making them available for the treatment of myocardial regeneration. However, although this is a possibility, stem cells do not turn into myocardial blasts to show significant improvement in function after infarction. In addition, there are reports of transplantation of progenitor cells, such as embryonic stem cells, into teratoma. Accordingly, studies have been conducted to differentiate adult stem cells such as bone marrow stem cells into cardiomyocytes and use them for transplantation.

Bone marrow stem cells are treated with cardiomyocytes by treating 5-azacytidine, TGF-beta1, insulin, and Hepatocyte Growth Factor, which are dimethylating agents. It has been reported that it can differentiate. However, 5-azacytidine randomizes genomic sequences to allow normal silent genes to be expressed. When 5-azacytidine is treated, cardiomyocytes as well as many other types of cells ( For example, muscle cells (MyoD-positive), bone cells (osteocalcin-positive), adipocytes (PPAR-positive), and myocardial blasts (cardiac troponin I positive) are induced, thus making them meaningful to one cell type, myocardial blast cells. Differentiation does not seem reproducible. In addition, dimethylation reagents, chemicals such as 5-azacytidine, have not been proven safe for human use.

On the other hand, studies are underway to replace dead heart tissue using skeletal myoblasts or skeletal myocytes. This is to extract a part of the muscle tissue of the patient to isolate and multiply the myoblasts to transplant into the heart, these cells do not form an intercellular gap junction (risk of arrhythmias), there is a risk of causing arrhythmias. That is, in order for the heart to beat, cardiac myocardial cells are required to communicate with each other, and the material is moved through a gap junction formed between cells, so that the signal is transmitted in a constant direction. Electrical signaling does not occur and may cause arrhythmia.

In order to succeed in cell transplantation, it is important to maximize the engraftment rate of the cells after transplantation, and since the cells do not progress to the final differentiation than the cells that have already been differentiated, cells with division capacity are known to have good engraftment ability and adaptability after transplantation. In addition, the cardiomyocytes have a characteristic of moving consistently through the electrical signal with the surrounding tissues, in order to transplant the cells in the myocardium and to improve the cardiac function, the transplanted cells are well fused with the existing tissues and the same signaling system It is important to move to.

In view of the above problems, the present invention maintains the characteristics of stem cells which are developmentally committed but not fully differentiated and thus divide, so that they are engrafted at the transplantation site and transferred to cardiomyocytes through signal transmission with surrounding tissues. The aim was to find appropriate biological growth factors and conditions that lead to one cell type (eg myocardial blast) that can be finally differentiated. As a result, bone marrow stem cells are treated with biological growth factors and committed to cardiac lineage while maintaining the characteristics of the stem cells, and furthermore, the cells at the stage showing the best engraftment and survival rate when transplanted into the damaged myocardium. Was developed, and the bone marrow stem cells induced through these methods were transplanted into the myocardium of myocardial infarction rats, thereby confirming that the transplanted cells were completely differentiated into cardiomyocytes, thereby improving cardiac function. .

Korean Patent Publication No. 10-2005-0055823 (published June 14, 2005) by the present applicant discloses cardiac specificity before transplantation so that cells derived from bone marrow stem cells exhibit good engraftment and survival rate in the damaged myocardium. It describes a method of culturing in a suitable medium for a certain period of time, the present invention is intended to maximize the therapeutic effect after cell transplantation by presenting in more detail the characteristics of the cells induced by this method.

An object of the present invention is to induce the bone marrow stem cells of mammals, including humans to cardiomyocytes using biological growth factors, to maintain the characteristics of the stem cells intact, the best engraftment in the myocardium damaged myocardial stem cells Mammals diagnosed with abnormalities due to inadequate myocardial function, such as myocardial infarction, by developing a method to obtain cells that exhibit survival rates and eventually differentiate into cardiomyocytes and achieve cardiac function. To provide clinically appropriately derived bone marrow stem cells.

To achieve the above object, a method for producing a cell for transplantation into mammalian myocardial tissue of the present invention comprises the following steps:

(a) supplying bone marrow stem cells that are not immortalized;

(b) subcultured with the bone marrow stem cells while maintaining the characteristics of the bone marrow stem cells;

(c) in cardiac specific media that induces bone marrow stem cells to be committed to myocardial blast cells while maintaining the characteristics of the bone marrow mesenchymal stem cells, 80-90% of the cells are induced to cardiomyocytes Culturing until done; And

(d) collecting the cells of step (c) when said cultured cells express characteristic markers of bone marrow mesenchymal stem cells.

Here, the bone marrow stem cells may be derived from the mammal to be transplanted, and in the step (c), the culture is preferably performed for 1 hour to 21 days, and more preferably for 6 to 7 days.

In addition, the cardiac specific medium is a medium containing a biological growth factor bFGF (basic Fibroblast Growth Factor), BMP-2 (Bone Morphogenic Protein-2) and IGF-I (Insulin-like Growth Factor-1), bFGF , Concentrations of BMP-2 and IGF-1 are 1 to 200 ng / ml, respectively, where 2 ~ 20% of bovine serum, 1 to 1000 μM of L-ascorbic acid-2-PO ₄ , and 5 to 15 ng / ml of LIF. , And dexamethasone 1 to 200 nM.

Here, the cultured bone marrow stem cells are committed to myocardial blast cells, specifically expressing MEF2 protein, not MF-20, CD29 +, CD90 +, CD44 +, CD34, which are characteristic markers of bone marrow mesenchymal stem cells It is desirable to collect cells expressing CD45-.

Cell production method for transplantation into mammalian myocardial tissue according to the present invention, a method for inducing induced bone marrow stem cells in the damaged myocardium for a specific period of time to show the optimal engraftment and survival rate and finally differentiate into cardiomyocytes; In addition, the cells thus induced are transplanted into a mammalian myocardial infarction model and evaluated.

First, a method of inducing induced bone marrow stem cells for a specific period of time to achieve optimal engraftment and survival in the damaged myocardium and finally to differentiate into cardiomyocytes comprises the following steps:

(1) supplying bone marrow stem cells that are not immortalized;

(2) passaging the bone marrow stem cells to obtain a sufficient number of cells;

(3) culturing the cultured bone marrow stem cells in cardiac specific media containing biological growth factors for 1 hour to 21 days to induce cardiomyocytes;

(4) collecting and monitoring the state of induction of the cells of step (3) at specific time intervals; And

(5) collecting the cells of step (3) when 80-90% of the cells are committed to myocardial blast cells and retain the characteristics of myeloid mesenchymal stem cells.

Methods for evaluating such induced cells by implanting them into a mammalian myocardial infarction model include the following steps:

(1) creating a myocardial infarction mammalian model;

(2) isolating bone marrow stem cells from the mammal;

(3) passaging the bone marrow stem cells to obtain a sufficient number of cells;

(4) culturing the cultured bone marrow stem cells in cardiac specific media containing biological growth factors for 6-7 days;

(5) labeling and collecting the cells of step (4) when 80-90% of the cells are committed to myocardial blast cells and retain the characteristics of bone marrow mesenchymal stem cells;

(6) transplanting the myocardial blast cells into the mammal; And

(7) confirming that the transplanted cells are finally differentiated from the heart of the mammal into cardiomyocytes and the cardiac function is improved.

In the present invention, the bone marrow stem cells of mammals, including humans, are treated with biological growth factors and directly committed to cardiac lineage, and the induced bone marrow stem cells exhibit the best engraftment and survival rate in the damaged myocardium. To induce differentiation, the cells were treated in vitro for a certain period of time before injection, and the induced bone marrow stem cells were transplanted into the heart of a mammal with myocardial infarction to evaluate the change in condition. Myocardial blast cells induced by this method are much more clinically appropriate than those using chemical dimethylating agents such as 5-azacytidine, and are well grafted to the surrounding tissues and fuse with the surrounding tissues after transplantation. Differentiation into cardiomyocytes, the results showed that the cardiac function was significantly improved.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

According to the present invention, the cells to be transplanted may be actively proliferated or sufficiently differentiated to enable mutual electrical signaling with surrounding tissues. That is, the transplanted cells can be differentiated into cells expressing Connexin-43, which is expressed at the late stage of cardiomyocyte development in the transplanted cardiac tissue and forms cell membrane channels between the cardiomyocytes, thereby enabling the transmission of electrical signals. Loss of heart tissue can be replaced to significantly improve heart function.

Bone marrow stem cells can be specifically induced and used as a cardiomyocyte system that can be identified using cardiac specific markers. The markers include, but are not limited to, Nkx2.5, GATA4, myosin enhancing factor (MEF2), and serum response factor (SRF).

In addition, myeloid stem cells induced myeloid stem cells can be identified as having a phenotype of bone marrow mesenchymal stem cells. Markers used to confirm this include, but are not limited to, CD13, CD29, CD31, CD34, CD44, CD45, CD73, CD90, CD105, CD106, CD133, c-kit, and the like.

Identification of markers associated with the heart and bone marrow mesenchymal stem cells can be performed by any detectable method, for example reporter gene expression (LacZ induced by a heart specific promoter), RNA expression (RT-PCR, Northern blot, RNase). protection methods), or protein expression (immunofluorescence staining, western blot, flow cytometry) and the like, but are not limited thereto.

Cells transplanted using the method can be used at any stage of the myoblast differentiation process.

"Bone marrow mesenchymal stem cell or bone marrow stem cell (BMSC)" as used herein refers to bone marrow mesenchymal-derived stem cells without CD45. Bone marrow mesenchymal stem cells are also called bone marrow stem cells or bone marrow-derived multipotential progenitor cells.

"Cardiac cell" refers to differentiated heart cells (eg cardiomyocytes) or cells (eg, cardiomyocytes or cardiomyocytes) that have been determined to produce or differentiate into cardiac cells.

"Cardiomyocyte" is a muscle that expresses, contracts and does not proliferate a detectable amount of marker in the heart (e.g. alpha myosin heavy chain, cTnI, MLC2v, alpha heart actin, Cx43 in vivo). It means a cell.

By "cardimyoblast" is meant a cell that expresses, contracts, and proliferates a detectable amount of cardiac marker in the heart.

"Cardiomyogenic cell" means a cell that expresses a detectable amount of Csx / Nkx2.5 RNA or protein, shows no organized sarcoma structure or contraction, and does not express a detectable amount of myosin heavy chain protein. do.

"Cardiac specific" refers to the early stages of cardiac differentiation in which early cardiac transcription factors are expressed before finally differentiated markers are expressed, and "cardiac specific media" is bone marrow stem A medium containing bFGF, BMP-2 and IGF-1, which induce cells into cardiomyocytes.

"Transdifferentiation" refers to the differentiation of germ cells into cells of another species by the appropriate external environment.

“Commitment” refers to the characterization of autologous stem cells and treatment at the early stage of cardiac differentiation, where early cardiac transcription factors are expressed before finally differentiated markers are expressed.

Hereinafter, an Example demonstrates this invention in detail. However, the following examples are only examples of the present invention, and the scope of the present invention is not limited only to the following examples. In addition, in the present embodiment, bone marrow stem cells collected from rat bone marrow are used, but the present invention is not limited thereto, and bone marrow stem cells obtained from all kinds of mammalian species including humans, dogs, mice, monkeys, and the like. Similarly applies.

Example  1: incubated with biological growth factors Committed  Bone marrow stem cells BMSC Cell surface Of marker  Confirm

Bone marrow stem cells can be roughly divided into two types of cells, hematopoetic stem cells and mesenchyma stem cells. Cell surface proteins of bone marrow stem cells in rats include CD34, CD45, and CD133 as hematopoietic stem cell markers, and CD29, CD90, CD44, and CD105 as mesenchymal stem cells, and are generally positive for c-kit. It is known. However, although cell surface proteins have different characteristics among murines, the characteristics of the cell surface proteins are not fully established, but hematopoietic stem cells are usually CD45 + and mesenchymal stem cells are CD45-, CD90 +, CD29 +, and CD44 +. Known.

Through the applicant's prior application, it was confirmed that bone marrow stem cells can be treated with growth factors and committed to myocardial blast cells. Thus committed bone marrow stem cells (committed BMSC) and bone marrow stems cultured without processing growth factors The characteristics of cell surface proteins were compared between cells (uncommitted BMSCs).

Isolate femoral stem cells of F344 rats to isolate internal bone marrow stem cells, followed by 10% bovine serum, 100M L-ascorbic acid-2-PO ₄ , 5-15 ng / ml mouse leukemia inhibitory factor (mLIF) and 20 nM dexamethasone. Incubated together. Under these in vitro conditions, bone marrow stem cells proliferate through subcultures without losing their reactivity to differentiation reagents such as growth factors. All incubators and slides were coated with collagen (5 ng / ml) at room temperature for 15 minutes before adding bone marrow stem cells. These in vitro conditions are used to ensure that bone marrow stem cells do not lose reactivity to differentiation agents such as growth factors by self-renewing and passage. In addition, bone marrow stem cells cultured through multiple passages maintain mesenchymal morphology and normal karyotype.

Induction of bone marrow stem cells into myocardial blast cells, after 2 passages (passage 2), 2-20% bovine serum in DMEM, 1-1000 μM L-ascorbic acid-2-PO ₄ , 5-15 ng / Committed bone marrow stems treated with medium containing mL mLIF, 1-200 nM dexamethasone, 1-200 ng / mL bFGF, 1-200 ng / mL BMP2, and 1-200 ng / mL IGF-I for 6-7 days Cells were produced. Uncommitted bone marrow stem cells not treated with biological growth factors were incubated with 10% bovine serum, 100M L-ascorbic acid-2-PO ₄ , 5-15 ng / ml mouse leukemia inhibitory factor (mLIF) and 20 nM dexamethasone It was. In addition, the bone marrow stem cells of rats were isolated with another control group and cultured with 10% bovine serum, 100M L-ascorbic acid-2-PO ₄ , 5-15 ng / ml mLIF, and 20 nM dexamethasone to grow to the bottom of the incubator. Lobe stem cells (Passage 0) were used.

The cell surface protein staining method is as follows: Cells were separated by 0.05% trypsin-EDTA. Separated cells 1 × 10 ⁵ / wherein the concentration of 100 μL - rat -CD29-FITC, anti-rat -CD90-FITC (green) and anti-rat -CD44-FITC, anti-rat -CD34-PE, wherein Rat-CD45-FITC (above direct staining), anti-rat-CD133, anti-rat-CD105, and anti-rat-c-kit (above indirect staining) were each treated with 10 ng. The reaction was carried out at 4 ° C. for 30 minutes and washed once with PBS. PBS was removed and samples of direct staining were refrigerated and fixed in 1% paraformaldehyde. Indirect samples, CD105 and c-kit, were added with Goth-anti-rabbit-Alexa 488-antibody. -Got-Alexa 488-antibody was added to react for 30 minutes at 4 ℃, PBS was added once washed and centrifuged. PBS was removed and the samples were refrigerated with 1% paraformaldehyde. Prepared samples were analyzed by FACS Calibur (BD, USA).

Figure 1 is a picture of analysis of bone marrow stem cell surface protein of Passage 0, Figure 2 is a picture of analysis of uncommitted bone marrow stem cell surface protein of Passage 2, Figure 3 is treated with growth factors in the bone marrow stem cells of Passage 2 6-7 days One committed bone marrow stem cell surface protein analysis picture.

As shown in the cell surface protein staining experiment in FIG. 1, bone marrow stem cells in passage 0 after bone marrow stem cell culture showed strong positive responses to CD90 (A), CD29 (B), CD44 (C) and CD45 (F). CD105 (D), CD34 (E), CD133 (G) and c-kit (F) were negative. In FIG. 2, uncommitted bone marrow stem cells of passage 2 (two passages) showed strong positive responses to CD90 (A), CD29 (B), and CD44 (C), and CD105 (D) and CD34. (E), CD45 (F), CD133 (G) and c-kit (F) was negative, even in the case of committed bone marrow stem cells of Figure 3 did not show a big difference.

From the above results, it can be seen that the bone marrow stem cells committed to myocardial blast cells treated with growth factors are still cells that have not lost the properties of the bone marrow stem cells.

Example  2: Rat  Myocardial blast ( CMC , cardiomyocyte Through co-culture with Committed  Committed bone marrow-derived mesenchymal  final differentiation of stem cells into myoblasts

Bone marrow stem cells were isolated from the femur of F344 rats and cultured with 10% bovine serum, 100M L-ascorbic acid-2-PO ₄ , 5-15 ng / ml mLIF (mouse leukemia inhibitory factor) and 20 nM dexamethasone.

Differentiation of bone marrow stem cells, after two passages, 2-20% bovine serum in DMEM, 1-1000 μM L-ascorbic acid-2-PO ₄ , 5-15 ng / ml mLIF, 1-200 nM dexamethasone, Medium containing 1-200 ng / ml bFGF, 1-200 ng / ml BMP2, and 1-200 ng / ml IGF-I was used. Bone marrow stem cells cultured under these conditions induce the expression of MEF2 protein, a marker protein of cardiomyocytes, is described in the applicant's prior application (Korean Patent Publication No. 10-2005-0055823 “Celling of Cells for Cell Transplantation”. Production method ”).

In order to obtain myocardial blast cells of F344 rats used in the mixed culture, the hearts of newborn rats (2 days old) were separated, washed with PBS, and only the ventricular sections were chopped and treated with 0.05% trypsin-EDTA at 37 ° C. for 10 minutes. The supernatant was then placed in DMEM medium containing 10% bovine serum and stored at 4 ° C., and this procedure was repeated seven times. The collected supernatants were centrifuged and placed in 10% bovine serum / DMEM medium and incubated for 1 hour in a 37 ° C. cell incubator containing 5% carbon dioxide. After 1 hour of culture, only fibroblasts adhered to the bottom of the incubator, and myocardial cells did not adhere to the incubator and remained in the culture. The culture medium containing myocardial blasts was collected and centrifuged, washed with PBS (Phosphate Buffered Saline), incubated in DMEM medium without bovine serum for 24 hours, and then the medium was removed and 10% bovine serum / DMEM medium was added thereto. Here, the cells cultured with growth factors and committed to myocardial blast cells were mixed with the myocardial blast cells isolated from the rat heart at a ratio of 4: 1, respectively, and the incubators were cultured in a 37 ° C. cell incubator containing 5% carbon dioxide.

The most important proteins that can prove the differentiation into complete cardiomyocytes include Connexin-43, a protein marker that creates gap junctions between cardiomyocytes, enabling the simultaneous contraction of muscle cells and the movement of intercellular contacts. Cardiac Troponin I, which constitutes cardiac muscle tissue, is the most used ( Nature , Vol. 410, 701-704, 2001).

To examine the differentiation of bone marrow stem cells into cardiomyocytes by mixed culture, Connexin-43 (green) and cardiac Troponin I (red) antibody, which are specific markers indicating the final differentiation of cardiomyocytes after 2 days of mixed culture, were used. Immunostaining was performed.

Figure 4 is a mixed culture of uncommitted bone marrow stem cells (A-D) and committed bone marrow stem cells (E-H) with myocardial blast cells for 2 days, the cardiomyocyte specific markers Connexin-43 (green) and cardiac Troponin I ( It is a fluorescence micrograph (400x) which measured the differentiation of bone marrow stem cells by staining red). 4, the mixed culture of uncommitted bone marrow stem cells (A to D) shows a very low frequency of cardiac Troponin I positive reaction with Connexin-43, a cardiomyocyte specific marker, but mixed with committed bone marrow stem cells. When cultured (E-F), Connexin-43 and cardiac Troponin I were positive in almost all cells. From these results, it can be seen that the committed myeloid stem cells prepared by the method of the present invention can be finally differentiated into cardiomyocytes present in the heart through mixed culture with cardiomyocytes, and their differentiation ability is greatly improved. have.

Example  3: Committed  Bone marrow stem cells Gap cushion  Formation and Functional Verification

By the above experiment, it was confirmed that the committed myeloid stem cells were finally differentiated into myoblasts expressing Connexin-43 through the mixed culture with the myoblasts. Then, it was confirmed whether the newly formed gap junctions showed normal function.

In the mixed culture according to the experimental method described above, uncommitted bone marrow stem cells or committed bone marrow stem cells treated with growth factors for 6 to 7 days were stained with DiI (Molecular Probe) dye (red color) to stain the cytoplasm. Staining in advance, at the same time the cells were cultured by treating the cells with Calcein AM (colored in green), which can be moved through a gap junction formed by Connexin-43. Two days after the mixed culture, the fluorescence microscope (Cal Zeiss Axio Butt 200) was observed to see whether Calcein AM stain of uncommitted bone marrow stem cells or committed bone marrow stem cells was transferred to rat cardiomyocytes. Then, to prove that the Calcein AM stain was shifted through the gap junction, heptanol, a specific substance that restricts the function of the gap junction, was added to the medium when the cells were cultured by the method described above. Observed after inhibiting migration

5 is a laser fluorescence micrograph (400 times) of the analysis of the function of the gap junction formed by Connexin-43 expressed in the mixed culture with myocardial blast cells, (A) ~ (C) is mixed with uncommitted bone marrow stem cells Cultured photographs, (D) to (F) are mixed cultured with committed bone marrow stem cells, (G) to (L) is treated with heptanol in each group to inhibit the gap junction by Connexin-43 It is a photograph observing the movement of Calcein AM stain.

In FIG. 5, C (uncommitted bone marrow stem cells) and F (committed bone marrow stem cells) are orange bone marrow stem cells, and green fluorescence is myocardial blast cells in which Calcein AM stain is transferred by gap junction. . 5 (A) to (F), the movement of the substance was not observed in the cells cultured with the uncommitted bone marrow stem cells, but the movement of the material to the myocardial blast cells co-cultured with the committed bone marrow stem cells. It was confirmed that it was active. In FIG. 5, (G) to (I) are cases of mixed culture of uncommitted bone marrow stem cells, and (J) to (L) are cases of mixed culture of committed cells. No movement of Calcein AM stain was observed in both groups.

According to the results of the above experiment, the growth factor-committed bone marrow stem cells are more differentiated into myocardial blast cells by mixed culture than the bone marrow stem cells that do not, and also form a gap junction with normal mass transfer function. It was confirmed.

Example  4: Weston Blurring  Myoblasts of Bone Marrow Stem Cells Commitment  Confirm

Bone marrow stem cells isolated from the femur of rats were expanded through passage culture. After two passages, the cells are divided into eight incubators, four of which are cultured with 10% bovine serum, 100M L-ascorbic acid-2-PO ₄ , 5-15 ng / ml mLIF (mouse). leukemia inhibitory factor) and a medium containing 20 nM dexamethasone, the other four in DMEM, myocardial cell differentiation medium, 2-20% bovine serum, 1-1000 μM L-ascorbic acid-2-PO ₄ , 5-15 ng / ml mLIF. A medium comprising 1-200 nM dexamethasone, 1-200 ng / ml bFGF, 1-200 ng / ml BMP2, and 1-200 ng / ml IGF-I was used.

Each culture medium was changed to fresh medium every 2 to 3 days, and cells were collected at 1, 3, 5 and 7 days after the medium treatment. The collected cells were extracted with a protein extraction reagent (Novagen). The extracted protein was electrophoresed for 2 hours at 40 mA using a 10% acrylamide gel. After electrophoresis, the gel was transferred to PVDF membrane and subjected to western blot.

Western blot detection of myocardial cell specific proteins Nkx2.5, MEF2 and GATA-4 proteins was as follows: Transfer the PVDF membrane to which the protein was transferred using a blocking solution (5% skim milk, 0.05% Tween-20). Trisma buffer solution) was blocked for 4 hours at room temperature, and washed three times for 15 minutes at room temperature with a wash solution (Trizma buffer solution containing 0.05% Tween-20). Goat-anti-Nkx2.5 (1: 200, SC-8697, Santa Cruz Biotechnonogy, Santa Cruz) antibody, rabbit-anti-MEF2 (1: 200, SC-10794, Santa Cruz Biotechnonogy, Santa Cruz) antibody, And rabbit-anti-GATA-4 (1: 200, SC-9053, Santa Cruz Biotechnonogy, Santa Cruz) antibody was added and reacted for 12 hours at 4 ℃. After the reaction, the membrane was washed three times with a washing solution for 15 minutes, and a secondary antigen (Hk (2.5 for Goth, MEF2 and GATA-4) for rabbit) with HRP (horse radish peroxidase) was added at 1: 10,000 for 1 hour. It was reacted at room temperature for a while. Thereafter, the membrane was washed three times with a washing solution for 15 minutes, and the washing solution was completely removed, followed by addition of an HRP substrate solution. After the reaction, the membrane was exposed to an X-ray film to analyze protein expression.

Figure 6 is a photograph showing the expression of the cardiomyocyte specific markers Nkx2.5, GATA-4, and MEF2 protein through Western blot experiment for uncommitted bone marrow stem cells and committed bone marrow stem cells, the fifth line of the rat It is a protein extracted from the heart and used as a positive control. As shown in FIG. 6, the committed myeloid stem cells cultured using the biological growth factor did not express the Nkx2.5 and GATA-4 proteins, which are myocardial blast specific markers, on day 1 and day 3 but began to appear from day 5 On the seventh day, the phenomenon was further increased. However, uncommitted bone marrow stem cells that did not use biological growth factors showed little protein expression until 7 days. MEF2 protein was also confirmed to significantly increase the amount of protein expression in committed myeloid stem cells.

Example  5: Committed  Myocardial Infarction Model of Bone Marrow Stem Cells Rat  Transplantation and Differentiation Marker  Expression Evaluation of Proteins

Twelve weeks old, female F344 rats (200-250 g body weight, Daehan Biolink) were used, and myocardial infarction model was made by ligating the left coronary artery for 45 minutes to occlude the vessels and after 45 minutes to release the ligation. Infarctions stabilized for 1 week. In addition, F344, an inbred rat, was used to prevent immune rejection.

The transplanted bone marrow stem cells were isolated from the femur of F344 rats, washed twice with DMEM medium and cultured medium (2-20% bovine serum, 1-1000 M L-ascorbic acid-2-PO ₄ , 1-200 nM dexamethasone). , And passage twice using 5-15 ng / ml mLIF), followed by 2-20% bovine serum in DMEM, 1-1000 μM L-ascorbic acid-2-PO ₄ , 5-15 ng / ml mLIF, Incubated for 6-7 days using medium containing 1-200 nM dexamethasone, 1-200 ng / ml bFGF, 1-200 ng / ml BMP2, and 1-200 ng / ml IGF-I and committed to cardiomyocytes Cells were produced. Uncommitted bone marrow stem cells were produced using culture media that did not contain biological growth factors. At the time of cell collection, cells were labeled with DiI, a red fluorescent label, to track the survival and differentiation of the cells after transplantation. Approximately 1 × 10 ⁶ cells were collected and a No. 26 syringe was placed at 6 to 10 infarcts of the heart. By injection. As a control group, myocardial infarction rats were injected with DMEM instead of cells. As another control group, rats that underwent chest incision without causing myocardial infarction were used.

After 8 weeks of cell transplantation, the heart was removed, and the tissue was attached to the glass slide using a frozen tissue section technique. DiI was measured for survival of the transplanted cells by fluorescence microscopy. In order to analyze the bone marrow stem cells transplanted into the heart tissue of myocardial infarction rats, the immune tissue antibody response was investigated.

7 is a picture showing the differentiation into myocardial blast cells through the immunostaining method after 8 weeks after injection of committed bone marrow stem cells into the heart of myocardial infarction rat, the red one shows the committed bone marrow stem injected by staining with DiI stain solution. Cells, green indicates myocardial blast specific markers Connexin-43 (A, B; 200-fold), cTnI (C-D; 200-fold), and MF-20 (E; 400-fold), F being the respective A graph showing the frequency of rats expressing marker proteins, gray for uncommitted and black for committed bone marrow stem cells. As shown in Figure 7, the committed bone marrow stem cells can be seen that when injected into the infarction myocardial engraftment and differentiation into myocardial blast cells. The ratio of this differentiation is shown in the graph of (F) of Figure 7, Connexin-43 was about 12% in rats injected with uncommitted bone marrow stem cells, about 60% in committed bone marrow stem cells 5 times higher, cTnI was more than 2 times higher at 32% and 75%, and MF-20 was 4 times higher at 12% and 50%, respectively.

From the above results, it can be seen that the rate of differentiation in vivo increased significantly compared to uncommitted bone marrow stem cells treated with growth factors.

Example  6: improving myocardial infarction

The rat myocardial infarction model was used to measure recovery recovery following bone marrow stem cell transplantation using echocardiography. Echocardiography was performed before cell transplantation, 4 weeks after transplantation, and 8 weeks after transplantation.

All 51 rats used in the experiment (10 sham operations, 14 medium injections, 15 uncommitted bone marrow stem cell injections and 12 committed bone marrow stem cells) were used.

8 is an 8-week echocardiogram of uncommitted bone marrow stem cells (A) and committed bone marrow stem cells (B) implanted in the heart of myocardial infarction rats, showing the end- and end-stem axial distances of the rats. (C) is a graph comparing left ventricular ejection fraction (LVEF) and (D) left ventricular fractional shortening (LVFS). From Figure 8 (A), the heart of rats injected with uncommitted bone marrow stem cells after myocardial infarction is shown that the inner axial distance between the left ventricular systolic end and the diastolic end is widened so that the function of the heart is inhibited due to myocardial infarction. have. On the other hand, from (B) of Figure 8, the heart of the rat injected with committed bone marrow stem cells can be seen that the internal axis distance of the left ventricular systolic end and dilator end is reduced. Therefore, it can be seen that the committed myeloid stem cells were finally differentiated into myocardial blast cells in the transplanted myocardial infarction site, thereby replacing the infarcted myocardium, thereby improving the contractile function of the heart.

In addition, both groups in which the left ventricular ejection fraction (LVEF) in FIG. 8 (C) and the left ventricular fractional shortening (LVFS) in (D) were injected with the medium alone could numerically indicate myocardial function. Even better, the myocardial function of rats injected with committed bone marrow stem cells showed a much better improvement.

From the above results, it can be seen that the contractile force of the infarct region in each animal is improved to harmonize with the adjacent region of the myocardial layer. The echocardiography results functionally reaffirm the tissue immunostaining results of Example 4 and demonstrate that transplantation of committed bone marrow stem cells cultured with growth factors significantly restores heart tissue after infarction.

Example  7. Improvement of arrhythmia

Death due to myocardial infarction is the main cause of the contraction of the heart and the inability to supply blood to the body. At the same time, arrhythmia caused by myocardial infarction may cause a heart attack by inhibiting simultaneous contraction due to abnormal heart muscle. Therefore, after bone marrow stem cells are transplanted, cell junctions with surrounding living cardiomyocytes should be freely established through gap junctions to prevent arrhythmia ( Nature , Vol. 410, 701-705, 2001). .

Rat myocardial infarction model was prepared according to the method described in Example 4 and injected with 1 × 10 ⁶ uncommitted bone marrow stem cells and committed bone marrow stem cells. Eight weeks after cell injection, anesthesia was injected into the femoral vessels and allowed to stabilize for 30 minutes. Thereafter, artificially induced aconitine (aconitine) was injected at a rate of 0.1 ml / min for 8 minutes and 30 seconds at a concentration of 10 mg / l. Arrhythmia was recorded by echocardiography for 20 minutes after aconitine injection, counting premature ventricular contractions (PVC's), and the incidence of ventricular tachycardia.

9 is a result of measuring the degree of arrhythmia induced by injecting an arrhythmia-inducing substance in the rat injected with bone marrow stem cells, (A) shows the incidence of ventricular tachycardia, (B) shows early ventricular contraction. As shown in Figure 9, ventricular premature contraction (B) occurred 2,205 ± 414 times in rats injected with uncommitted bone marrow stem cells, while significantly reduced to 655 ± 302 cycles in rats injected with committed bone marrow stem cells. In addition, in the incidence of ventricular tachycardia (A), the rats injected with committed myeloid stem cells were lower at 75% and 67%, respectively.

From the above results, committed myeloid stem cells cultured with biological growth factors are finally differentiated into myoblasts at the transplantation site, and in harmony with the myocardium at the periphery of the myocardial infarction, the heart contraction, thus risking arrhythmia. It can be seen that can be reduced.

The cells produced according to the method of the present invention are committed to myocardial blast cells while maintaining the characteristics of stem cells, and when transplanted into the heart, finally differentiate into cardiomyocytes capable of mutual electrical signal transmission with existing peripheral tissues. Clinically appropriate for According to the method of the present invention, it provides clinically optimized engraftment and survival rate, and provides committed bone marrow stem cells that can fully function as cardiomyocytes and improve cardiac function, and diagnosed with abnormalities due to insufficient myocardial function. It can be used to treat mammals (eg, humans).

Claims (9)

(a) supplying bone marrow stem cells that are not immortalized; (b) subcultured with the bone marrow stem cells while maintaining the characteristics of the bone marrow stem cells; (c) in cardiac specific media that induces bone marrow stem cells to be committed to myocardial blast cells while maintaining the characteristics of the bone marrow mesenchymal stem cells, 80-90% of the cells are induced to cardiomyocytes Culturing until done; And (d) collecting the cells of step (c) when said cultured cells express characteristic markers of bone marrow mesenchymal stem cells, Method of producing cells for transplantation into mammalian myocardial tissue. The method of claim 1, wherein said bone marrow stem cells are derived from a mammal to be transplanted. The method of claim 1, wherein the mammal is a human. The method of claim 1, wherein the step (c) is incubated for 6-7 days. The method of claim 1, wherein the cardiac specific medium is a medium comprising a biological growth factor. 6. The method of claim 5, wherein the biological growth factors are basic Fibroblast Growth Factor (bFGF), Bone Morphogenic Protein-2 (BMP-2) and Insulin-like Growth Factor-1 (IGF-I). The method according to claim 6, wherein the concentration of bFGF, BMP-2 and IGF-1 is 1 to 200 ng / ㎖, 2 to 20% of bovine serum, L- ascorbic acid-2-PO ₄ 1 to 1000 μM, LIF 5-15 ng / ml, and dexamethasone 1-200 nM. The method of claim 1, wherein the cells of the step of expressing MEF2 protein and not expressing MF-20 are collected. The method of claim 1, wherein the characteristic markers of bone marrow mesenchymal stem cells are CD29 +, CD90 +, CD44 +, CD34−, CD45−.

Images