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An agent for differentiating hematopoietic stem cell into natural killer cell comprising YC-1 or IL-21 and a method of differentiating hematopoietic stem cell into natural killer cell using thereof

A technology of IL-21 and hematopoietic stem cells, applied in the field of natural killer cells, can solve the problem that the mechanism of YC-1's anti-cancer activity has not been disclosed.

Inactive Publication Date: 2010-08-11
KOREA RES INST OF BIOSCI & BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although YC-1 is very useful for studying HIF-1α (Moeller BJ, et al., Cancer cell 5:429-441, 2004; Funasaka T, et al., FASEB J, 19:1422-1430, 2005; Sun.H.L., et al., Oncogene 26:3941-3951, 2007), but the mechanism of YC-1 anticancer activity has not yet been disclosed (Sun.H.L., et al., Oncogene 1-11, 2006)

Method used

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  • An agent for differentiating hematopoietic stem cell into natural killer cell comprising YC-1 or IL-21 and a method of differentiating hematopoietic stem cell into natural killer cell using thereof
  • An agent for differentiating hematopoietic stem cell into natural killer cell comprising YC-1 or IL-21 and a method of differentiating hematopoietic stem cell into natural killer cell using thereof
  • An agent for differentiating hematopoietic stem cell into natural killer cell comprising YC-1 or IL-21 and a method of differentiating hematopoietic stem cell into natural killer cell using thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Example 1: Isolation of hematopoietic stem cells from umbilical cord blood

[0092] The umbilical cord blood was provided by the Obstetrics and Gynecology Department of Konyang University Hospital in Daejeon, Korea. Since it was only used for research, it was only diluted with RPMI 1640 (GIBCO-BRL, USA) at a ratio of 2:1. The prepared cord blood was loaded on top of Ficoll-Paque (Sigma, USA), followed by centrifugation (20,000 rpm, 30 minutes) to separate mononuclear cells (MNC). Red blood cells were removed from the harvested cells and the harvested monocytes were labeled with hematopoietic stem cell marker 'CD34 magnetic beads'. Next, CD34+ cells were isolated using MS / RS column and MACS (Magnetic Activated Cell Sorting). The obtained CD34+ hematopoietic stem cells (hereinafter referred to as "HSC cells") were measured to have a purity of 92% ( figure 1 ).

Embodiment 2

[0093] Example 2: Differentiation of umbilical cord blood hematopoietic stem cells to NK cells

[0094] The HSC cells isolated from umbilical cord blood in Example 1 were inoculated into 12-well plates (Falcon, USA), and the wells contained 30ng / ml human factor (PeproTech, USA), 50ng / ml human Flt3L (FMS-like tyrosine acid kinase 3 ligand, PeproTech, USA), 5ng / ml human IL-7 (PeproTech, USA), and 10 -6 M water-based (hydro) SCF (stem cell hydrocortisone, Stem cell Technology, CA) Myelocult complete medium (Stem cell Technology, CA), 1 × 10 per well 6 cells, followed by incubation at 37°C, 5% CO 2 cultured in an incubator for 14 days. After 3 days, half of the supernatant was replaced with fresh medium containing cytokines with the same composition as above. In order to differentiate into mature NK cells (hereinafter referred to as "NK cells"), HSC cells were recovered after 14 days and cultured for another 14 days in the presence of human IL-15 (30 ng / ml, PeproTech, USA). ...

Embodiment 3

[0095] Example 3: Effect of IL-21 on NK cell differentiation

[0096] In order to investigate the effect of IL-21, which is known to promote differentiation and increase activity, on the differentiation of NK cells, during the differentiation process of umbilical cord blood hematopoietic hepatocytes into mature NK cells (mNK) via NK precursor cells (pNK) IL-21 (20ng / Ml, PeproTech, USA) was processed. Cells were cultured in the presence of IL-15, followed by FACS analysis and 51 Cr release analysis.

[0097] IL-21 has the amino acid sequence shown in SEQ.ID.NO:1, and the nucleotide sequence is shown in SEQ.ID.NO:2.

[0098] From the results of FACS analysis, it can be determined that the number of CD56+NK cells in the IL-21 treatment group increased ( Figure 4 and 5 ).

[0099] From 51 As a result of Cr release analysis, it can be confirmed that the killing activity of NK cells treated with IL-21 increased ( Figure 6 ). E:T (effector cells: target cells) was 2.5:1. ...

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Abstract

The present invention relates to an agent for differentiating hematopoietic stem cells into natural killer cells and a method for the differentiation, more precisely an agent for differentiating hematopoietic stem cells into natural killer cells comprising YC-I [3- ( 5'-hydroxymethyl-2'-furyl)-1-benzylindazole] or IL-21 (Interleukin-21) as an active ingredient and a method for differentiating hematopoietic stem cells into natural killer cells using the same. The YC-I and IL-21 regulate the differentiation of hematopoietic stem cells into natural killer cells and increase the killing activity of NK cells. Therefore, the agent for NK cell differentiation of the present invention can be effectively used for cell therapy for the treatment of cancer by regulating the differentiation of hematopoietic stem cells into natural killer cells having tumor cell killing activity.

Description

technical field [0001] The present invention relates to a medicament for differentiating hematopoietic stem cells into natural killer cells and a method thereof, more particularly to a method for differentiating hematopoietic stem cells into natural killer cells containing YC-1[3-(5'-hydroxymethyl -2'-furan)-1-benzyl indazole] or IL-21 (interleukin-21) as an active ingredient and a method for differentiating hematopoietic stem cells into natural killer cells using the same. Background technique [0002] Hematopoietic stem cells are a type of adult stem cells that have the potential to differentiate into almost all hematopoietic cells (including red blood cells, white blood cells, platelets and lymphocytes), and can continuously and automatically regenerate from bone marrow hematopoietic stem cells as immune cells. Cells forming the immune system, particularly natural killer cells (hereinafter referred to as "NK cells") have the ability to kill tumor cells non-specifically. ...

Claims

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Application Information

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IPC IPC(8): A61K31/416
CPCA61K31/416A61P35/00A61K31/404
Inventor 崔仁杓尹锡兰尹沼贤郑秦雄
Owner KOREA RES INST OF BIOSCI & BIOTECH
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