KR101453645B1 - Pharmaceutical composition for treating metabolic disease comprising human blood derived hematosphere - Google Patents

Abstract

The present invention relates to a method for efficiently differentiating into insulin-secreting cells using human blood-derived blood cell mass (BBHS), a therapeutic agent for metabolic diseases using the same, and the like. It is expected that the development of a therapeutic agent for metabolic diseases using the differentiated insulin-secreting cells according to the present invention will be put to practical use. By using the hematopoietic stem cells derived from human blood according to the present invention, Development, immune rejection, ethical problems, and differentiation methods.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for treating metabolic diseases including hematopoietic cells derived from human blood,

The present invention relates to a method for efficiently differentiating into insulin-secreting cells using human blood-derived blood cell mass (BBHS), a therapeutic agent for metabolic diseases using the same, and the like.

Stem cells are pluripotent cells, which can be differentiated into any cell. Stem cells are basically composed of Embryonic Stem Cell (ESC), Induced Pluripotent Stem Cell (IPS) and Adult Stem Cell (ASC) (AS Boyd, KJ Wood, Characteristics of the early immune response following transplantation of mouse ES cell-derived insulin-producing cell clusters, PLoS One, 2010). However, there are various problems at present. For example, embryonic stem cells (ESCs) are proliferating and capable of differentiating into all cells, but the ongoing proliferation has not solved the problem of tumor development, immune rejection, and ethical problems. In order to overcome the problems of ESCs, degenerative stem cells (IPS) have been developed in 2006 (Kazutoshi Takahashi and Shinya Yamanaka, Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell, 2006; 126: 663-676), there are various problems to be used for actual treatment, for example, viruses and immune rejection. In addition, adult stem cells (ASC) have few cells, proliferation is not active, and there is a difficulty in the method of differentiating into insulin-secreting cells.

The present invention relates to the development of a stem cell therapeutic agent using previously developed ESCs, degenerated stem cells (IPS) and adult stem cells (ASC), and the like in the fields of tumor development, immune rejection, ethical problems, (BBHS) to induce differentiation into insulin-secreting cells, thereby providing a method of using the same as a therapeutic agent for metabolic diseases.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to solve the above problems, the present invention provides a pharmaceutical composition for the treatment of metabolic diseases comprising hematopoietic cells derived from human blood.

In one embodiment of the present invention, the hematopoietic cell population derived from human blood is characterized by being formed through three-dimensional coagulation culture by separating mononuclear cells from human blood.

In another embodiment of the present invention, the hematopoietic cell derived from human blood is characterized in that it is induced into an insulin secretory cell.

In another embodiment of the present invention, the metabolic disease is characterized by being selected from the group consisting of diabetes, hyperlipidemia, and obesity.

The present invention also provides a method for treating a metabolic disease by administering to a subject a pharmaceutically effective amount of the pharmaceutical composition produced by the present invention.

According to the present invention, it is expected that insulin secretory cells can be differentiated using hematopoietic cells derived from human blood, and that the use of the insulin-secreting cells will contribute to the practical development of a therapeutic agent for metabolic disease cells. The use of hematopoietic stem cells derived from human blood according to the present invention solves the conventional problems associated with the development of stem cell treatment agents, namely, tumor development, immunodeficiency, ethical problems, differentiation methods and existing cells as well as being difficult to obtain Lt; / RTI >

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a view showing a method of forming a blood hematopoietic cell (BBHS) derived from human blood. FIG.
FIG. 2 shows a result of confirming that some cells expressing insulin were present in a human blood-derived blood cell mass using immunofluorescence staining (green: insulin, blue: nuclear).
FIG. 3 shows that Nestin is expressed in human blood-derived blood cell mass (BBHS) through immunofluorescence staining (green: Nestin, blue: nuclear, Scale bar: 50um).
FIG. 4 shows the results of beta-tubulin III expression in human blood-derived blood cell mass (BBHS) using immunofluorescent staining (green: beta-tubulin III, blue: nuclear, Scale bar: 50um).
FIG. 5 is a diagram showing a process of inducing insulin secretory cell differentiation using human blood-derived blood cell mass.
FIG. 6 shows the result of confirming genes important for insulin expression using the reverse transcription polymerase chain reaction (RT-PCR) technique at each step of inducing differentiation of insulin-secreting cells.
FIG. 7 shows that insulin (red) and Nestin (green) were stained by immunofluorescence staining after differentiation into human insulin-secreting cells from human blood hematopoietic cell mass (BBHS). As a result, most cells expressed insulin (Red: insulin, green: Nestin, blue: nuclear, Scale bar: 50um).
FIG. 8 shows the results of the differentiation of human blood-derived hematopoietic cells and insulin-secreting cells using a Dithizone staining technique which detects insulin and shows a red color. As a result, in step 4, which is the last stage of differentiation into insulin- (Scale bar: 10um).
9 is a graph comparing the glucose-stimulated insulin secretion (GSIS) of insulin-secreted cells derived from human blood-derived blood cell mass (BBHS) with insulin secretion in glucose at a high concentration to be.

The present inventors confirmed the formation of an insulin secretory cell-friendly microenvironment through the blood-born hematosome (BBHS) derived from human blood and the ability of mononuclear cells to differentiate into insulin-secreting cells. The present invention has the effect of promoting the survival and differentiation of insulin-secreting cells in stem cell therapy and gene therapy using human hematopoietic cell mass, and can be used for the development of a stem cell therapeutic agent for various metabolic diseases such as diabetes, hyperlipidemia or obesity It is expected to be.

Accordingly, the present invention provides a pharmaceutical composition for the treatment of metabolic diseases comprising hematopoietic cells derived from human blood. The hematopoietic cell derived from human blood is induced into insulin-secreting cells. The metabolic diseases include diabetes, hyperlipidemia, obesity and the like, but are not limited thereto as long as they are all diseases that are related to insulin secretion metabolic function.

The term " blood-born hematopoietic cells (BBHS) "used in the present invention refers to an aggregate of mononuclear cells existing in the blood and specific cells having stem cells, and forms a three- It means that it forms a spherical shape like the inner cell masses of aeration.

The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may include, but is not limited to, physiological saline, polyethylene glycol, ethanol, vegetable oil, and isopropyl myristate.

The present invention also provides a method for treating a metabolic disease by administering to said individual a pharmaceutically effective amount of said pharmaceutical composition. In the present invention, an 'individual' refers to a subject in need of treatment for diseases, and more specifically, a human or non-human primate, mouse, rat, dog, cat, horse, And the like. It is to be understood that the term "pharmaceutically effective amount" in the present invention can be variously adjusted depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, .

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the drug form, the administration route, and the period, but can be appropriately selected by those skilled in the art. However, it is preferably administered at a daily dose of 0.001 to 100 mg / kg body weight, more preferably 0.01 to 30 mg / kg body weight. The administration can be carried out once a day or divided into several times.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. The method of administration is not limited and can be administered, for example, orally, rectally, or by intravenous, intramuscular, subcutaneous, intrauterine, or intra-cerebroventricular injection.

The pharmaceutical composition of the present invention can be prepared into various pharmaceutical formulations, and there is no limitation on the form of the formulation.

The present invention has shown that a human blood-derived hematopoietic cell (BBHS) forms a blood-born hematosome (BBHS) (Example 1), and insulin is expressed in the blood hematopoietic cell (BBHS) Secretory cell differentiation was induced (Example 3). Finally, it was confirmed whether the insulin or the insulin was actually secreted and the gene important for insulin expression (Example 4).

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description of the present invention, a detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear. In addition, the preferred embodiments of the present invention will be described below, but it is needless to say that the technical idea of the present invention is not limited thereto and can be variously modified by those skilled in the art.

Example 1 describes the process of forming blood-born hematosome (BBHS) from human blood, and Example 2 describes the characteristics of hematopoietic cell derived from human blood. Example 3 described the process of inducing insulin secretory cell differentiation in human blood-derived blood cell mass (BBHS). In Example 4, it was confirmed whether genes actually important for insulin or insulin expression and insulin were actually secreted.

Example  1. Human hematopoietic stem cells Cell mass  ( blood - born hematosphere , BBHS ) ≪ / RTI >

(1) Mononuclear cell separation step in peripheral blood

Peripheral blood was obtained by applying Heparin (approximately 100 ul) to a 50 ml syringe. 10 ml of peripheral blood was poured into 50 ml tubes, and 30 ml of PBS (phosphate-buffered saline) was added and carefully mixed. 10 ml of Ficoll used for the density gradient in the diluted blood was slowly poured into the bottom of the tube using a pipette to separate the clear Ficoll layer and the red blood layer and then the centrifugation was performed at 2500 rpm at 25 ° C for 30 minutes, Respectively. After the yellow serum layer, white mononuclear cell layer and transparent Ficoll layer were separated at the upper part, red erythrocytes and mononuclear layer were separated. Then, the upper yellowish layer was inhaled and the white mononuclear cell layer was carefully transferred to a new tube. The transferred mononuclear layer was divided into two tubes, filled with PBS, and centrifuged at 1,800 rpm at 4 ° C for 10 minutes. The cell pellet was vortexed and unfolded into single cells, filled to the end with PBS, and centrifuged at 1700 rpm for 10 min at 4 ° C. The above rinse procedure was repeated three times to remove the substances used in the density gradient and the residues in the blood. Cell counts were measured with a hemocytometer prior to the final centrifugation.

(2) Three-dimensional culture step

The rinsed cells were suspended in an ultra-low attach culture dish at a high density of 10 ⁶ / ml or more using a culture medium supplemented with 5% FBS in Endothelial basal medium-2 (EBM-2) After incubation at 37 ° C in a 5% CO ₂ -concentrated incubator, the same medium was added for the first two days after incubation.

FIG. 1 is a schematic diagram showing a process of culturing a blood-born hematosphere (BBHS) for 5 days after the separation of human peripheral blood mononuclear cells (PBMC).

(3) Cell mass  Disassembly step into single cell

The cell mass obtained through the culturing process was separated into single cells for use in characterization and treatment. The suspended cells were collected by centrifugation at 4 ° C for 10 minutes at 1,700 rpm. After the culture plate was shaken horizontally and vertically, the cells were collected in the center. The cell pellet was lightly loosened with 1 ml of Accutase, a cell disassembly solution, and cultured in a 37 ° C incubator for 2 to 3 minutes. After incubation, 1 ml of the cell culture was added, followed by several pipetting, followed by centrifugation at 1,700 rpm at 4 ° C for 10 minutes.

Example  2. Human hematopoietic stem cells In the cell mass  Identification of insulin-secreting cells

In FIG. 2, in order to confirm the expression level of insulin in human blood-derived hematopoietic cells developed by the present invention, immunofluorescence was used to stain insulin. As a result, cells expressing some insulin (Nestin and beta-tubulin ).

For immunofluorescence staining, human blood-derived hematopoietic cells were fixed for 30 minutes, washed three times with PBS, blocked for 30 minutes, and stained with insulin antibody (green: insulin, blue: nuclear ).

Nestin and beta-tubulin-expressing neuronal progenitor cells and neurons and insulin precursors are known to share many parts and to be easily differentiated into insulin secretory cells (IPC) (Hori Y, Gu X, Xie X , Kim SK. Differentiation of insulin-producing cells from human neural progenitor cells.

Therefore, the expression level of Nestin and beta-tubulin in human blood-derived hematopoietic cells was analyzed using a confocal microscope using immunofluorescence staining method using the Nestin and beta-tubulin antibodies in the same manner as in Fig.

As a result, it was demonstrated that there are many cells expressing Nestin (see FIG. 3) and beta-tubulin (see FIG. 4) in human hematopoietic cell mass (FIG. 3 - green: Nestin, , Blue: nuclear, Scale bar: 50um).

Example  3. Human hematopoietic stem cells In the cell mass  Induction of insulin secretory cell differentiation

FIG. 5 shows a process of culturing a human hematopoietic cell line developed by the present invention for 7 days to differentiate into insulin-secreting cells.

More specifically, in the first step, blood cells derived from human blood were cultured for 7 days, and then human blood hematopoietic cells (BBHS) were transferred from 5 g / ml Fibronectin-coated culture dishes. EBM- Glucose (5.9 mM) for 2 days. In the second step, EBM-2 was changed to medium containing 1% FBS and high-glucose (25 mM) and cultured for 2 days. In the third step, EBM-2 was replaced with medium supplemented with 1% FBS and low-glucose (5.9 mM), N2 supplement (Invitrogen), and cultured for 2 days. Finally, in step 4, EBM-2 was replaced with medium supplemented with 1% FBS, High-Glucose (25 mM), and 10 mM Nicotinamide (Sigma Aldrich) and cultured for 2 days.

Example  4. Characterization of genes and proteins after induction of differentiation into insulin-secreting cells

CDNA was synthesized using Reverse Transcription Polymerase Chain Reaction (RT-PCR) at each step of differentiating the human blood-derived blood cell mass (BBHS) developed by the present invention into insulin-secreting cells, (GLUT2), insulin promoter factor 1 (Pdx-1), Neurogenin 3 (Ngn3), Nkx6.1, Proprotein convertase 2 (PC2), PC1 / 3, SUR1 and GAPDH, which are important genes for insulin and insulin secretion PCR was carried out using a primer specific for the gene. The PCR products were analyzed using agarose gel electrophoresis. The results of confirming the expression of these genes are shown in FIG.

In particular, the most important insulin genes were increased, insulin promoter factor 1 (Pdx-1), Neurogenin 3 (Ngn3), and Nkx6.1, which are known as transcription factor genes important for pancreatic and beta cell development Respectively. Glucose transporter 2 (GLUT2) and proprotein convertase 2 (PC2), which are important for the endocrine function of the pancreas, increased with each step. In addition, Kir6.2, an important function of beta cells, and ATP-binding cassette transporter sub-family C member 8 (SUR1) also increased with each step. The primers used at this time are shown in Table 1 below.

Primer Sequence Access Number Product size (bp) Insulin Forward 5'-CCTGTGCGGCTCACACCTGG-3 ' NM_000207 540 Reverse 5'-CCACTCAGGCAGGCAGCCAC-3 ' Glut2 Forward 5'-AGGACTTCTGTGGACCTTATGTG-3 ' L09674 231 Reverse 5'-GTTCATGTCAAAAAGCAGGG-3 ' Pdx1 Forward 5'-GGATGAAGTCTACCAAAGCTCACGC-3 ' NM_000209 220 Reverse 5'-CCAGATCTTGATGTGTCTCTCGGTC-3 ' Ngn3 Forward 5'-CGTGAACTCCTTGAACTGAGCAG-3 ' AF234829 211 Reverse 5'-TGGCACTCCTGGGACAGATTTC-3 ' Nkx6.1 Forward 5'-CAATGGAGGGCACCCGGCAG-3 ' NM_006168.2 599 Reverse 5'-CCAGAAGATGGGCGTCCGGC-3 ' PC2 Forward 5'-GCATCAAGCACAGACCTACACTCG-3 ' NM_002594 309 Reverse 5'-GAGACACAACCCTTCATCCTTC-3 ' PC1 / 3 Forward 5'-TTGGCTGAAAGAGAACGGGATACATCT-3 ' NM_000439 457 Reverse 5'-ACTTCTTTGGTGATTGCTTTGGCGGTG-3 ' SUR1 Forward 5'-CACATCCACCACAGCACATGG-3 ' NM_000352.3 420 Reverse 5'-GTCTTGAAGAAGATGTATCTCCTCA-3 ' GAPDH Forward 5'-CAAATTCGTTGTCATACCAG-3 ' NM_002046 480 Reverse 5'-CGTGGAAGGACTCATGAC-3 '

FIG. 7 shows the expression of insulin and Nestin by immunofluorescence staining after induction of insulin secretory cell differentiation in human hematopoietic stem cells (BBHS) through immunofluorescence staining. As a result, insulin and Nestin were expressed. In addition, when human blood hematopoietic cells were induced to differentiate into insulin-secreting cells, insulin-expressing cells were further increased (red: insulin, green: Nestin, blue: nuclear, Scale bar : 50um).

FIG. 8 shows that insulin secretion is detected by inserting zinc ions (zinc ions) in the insulin-secreting cells, and Dithizone staining (DTZ), which is stained with red (Crimson red) (Scale bar: 10um). For DTZ staining, each step of differentiation of insulin-secreting cells was stained by adding DTZ (100 ug / ml) to EBM-2 medium containing 1% FBS.

In FIG. 9, glucose-stimulated insulin secretion (GSIS) was assayed by enzyme-linked immunosorbent assay (ELISA) using supernatant of cells to confirm whether insulin secretion actually occurred Respectively.

More specifically, GSIS was washed once with PBS after induction of differentiation of insulin-secreting cells in human blood-derived blood cell mass (BBHS), and Krebs-Ringer bicarbonate (KRB) buffer (120 mM NaCl, 5 mM) containing low glucose mM KCl, 2.5 mM CaCl ₂ , 1.1 mM MgCl ₂ , 25 mM NaHCO ₃ , 0.1 g BSA) for 1 hour at 37 ° C. Then, the supernatant was removed, and then a new KRB buffer containing 5.9 mM (Low glucose) or 25 mM glucose (High glucose) was added to each well. After incubation at 37 ° C. for 2 hours, supernatant was obtained and enzyme-linked immunosorbent assay (Enzyme-linked immunosorbent assay, ELISA). As a result, it was confirmed that more insulin was secreted from glucose at a high concentration than at low concentration of glucose. That is, it was confirmed that insulin-secreting cells derived from human blood-derived blood cell mass (BBHS) secrete insulin by glucose stimulation (see FIG. 9).

It will be apparent to those skilled in the art that various modifications, substitutions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims. will be.

Therefore, the embodiments disclosed in the present invention and the accompanying drawings are intended to illustrate and not to limit the technical spirit of the present invention, and the scope of the technical idea of the present invention is not limited by these embodiments and the accompanying drawings . The scope of protection of the present invention should be construed according to the following claims, and all technical ideas falling within the scope of the same shall be construed as falling within the scope of the present invention.

<110> SNU R & DB FOUNDATION <120> PHARMACEUTICAL COMPOSITION FOR TREATING METABOLIC DISEASE          COMPRISING HUMAN BLOOD DERIVED HEMATOSPHERE <130> PB13-11179 <150> KR 2012/0006070 <151> 2012-01-19 <160> 18 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Insulin (NM_000207) Forward primer <400> 1 cctgtgcggc tcacacctgg 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Insulin (NM_000207) Reverse primer <400> 2 ccactcaggc aggcagccac 20 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Glut2 (L09674) Forward primer <400> 3 aggacttctg tggaccttat gtg 23 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Glut2 (L09674) Reverse primer <400> 4 gttcatgtca aaaagcaggg 20 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Pdx1 (NM_000209) Forward primer <400> 5 ggatgaagtc taccaaagct cacgc 25 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Pdx1 (NM_000209) Reverse primer <400> 6 ccagatcttg atgtgtctct cggtc 25 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Ngn3 (AF234829) Forward primer <400> 7 cgtgaactcc ttgaactgag cag 23 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Ngn3 (AF234829) Reverse primer <400> 8 tggcactcct gggacagatt tc 22 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nkx6.1 (NM_006168.2) Forward primer <400> 9 caatggaggg cacccggcag 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Nkx6.1 (NM_006168.2) Reverse primer <400> 10 ccagaagatg ggcgtccggc 20 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> PC2 (NM_002594) Forward primer <400> 11 gcatcaagca cagacctaca ctcg 24 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PC2 (NM_002594) Reverse primer <400> 12 gagacacaac ccttcatcct tc 22 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> PC1 / 3 (NM_000439) Forward primer <400> 13 ttggctgaaa gagaacggga tacatct 27 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> PC1 / 3 (NM_000439) Reverse primer <400> 14 acttctttgg tgattgcttt ggcggtg 27 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> SUR1 (NM_000352.3) Forward primer <400> 15 cacatccacc acagcacatg g 21 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> SUR1 (NM_000352.3) Reverse primer <400> 16 gtcttgaaga agatgtatct cctca 25 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH (NM_002046) Forward primer <400> 17 caaattcgtt gtcataccag 20 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GAPDH (NM_002046) Reverse primer <400> 18 cgtggaagga ctcatgac 18

Claims (5)

Claims 1. A pharmaceutical composition for the treatment of diabetes comprising an insulin secretory cell differentiation-inducing human blood-derived hemocyte cell mass,
Wherein the human hematopoietic cell population increases PDX-1, Ngn-3, Nkx6.1, PC2, or Sur-1 expression.
The method according to claim 1,
Wherein the human blood-derived hemocyte cell mass is formed through three-dimensional coagulation culture by separating mononuclear cells from human blood.
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