WO2007010586A1

WO2007010586A1 - Hematopoietic stem cell identifier

Info

Publication number: WO2007010586A1
Application number: PCT/JP2005/012984
Authority: WO
Inventors: Hideaki Nakajima; Toshio Kitamura; Yuko Koshino; Monica Lik-Shing Tsang; Fumi Shibata
Original assignee: The University Of Tokyo; Research And Diagnostics Systems, Incorporated
Priority date: 2005-07-14
Filing date: 2005-07-14
Publication date: 2007-01-25
Also published as: JPWO2007010586A1

Abstract

Disclosed are a hematopoietic stem cell identifier and a method for identifying a hematopoietic stem cell which can identify a hematopoietic stem cell directly, and a process for preparing a highly condensed fraction rich in hematopoietic stem cells. A hematopoietic stem cell identifier comprising an antibody which is produced using Robo-4 protein as an antigen and can specifically bind to the protein; a hematopoietic stem cell identifier comprising a Robo-4 gene; a method for identifying a hematopoietic stem cell using the identifier; and a process for preparing a highly condensed fraction rich in hematopoietic stem cells.

Description

Specification

Hematopoietic stem cell identification agent

Technical field

The present invention relates to an agent for identifying hematopoietic stem cells, a method for identifying hematopoietic stem cells, and a method for preparing a highly concentrated fraction of hematopoietic stem cells.

Background art

[0002] Stem cells are divided into embryonic stem cells and somatic stem cells. Embryonic stem cells have the ability to sort into all types of cells that make up the embryo, and have a proliferative capacity close to infinity. Somatic stem cells, on the other hand, have the ability to be divided into a certain cell lineage, among them the ability to multiply and self-proliferate.

[0003] A hematopoietic stem cell is a somatic stem cell that has the potential to produce all types of blood cells and the ability to self-replicate to continue to produce the same cells as itself. When bone marrow transplantation is performed, it refers to cells that can reconstitute all blood cell lineages over a long period of several months.

[0004] As an efficient method for concentrating hematopoietic stem cells, there is a method using FACS (fluorescence-activated cell sorter). As an index when using FACS, cell surface markers, fluorescent substance GFP (green fluorescent protein), and SP (side population) cells having the property of discharging Hoechst 33342, which is a dye that binds to DNA, are known. The

[0005] In FACS fractionation using cell surface markers, c Kit positive, Sea-1 positive and 1 ineage marker negative! /, A fraction with the expression pattern of a cell surface antigen (KSL fraction), Furthermore, CD34-- KSL and KSL-SP fractions with CD34-negative expression have been found to contain high concentrations of hematopoietic stem cells, and these fractions are often used synonymously with hematopoietic stem cells. Yes. However, these cell surface markers were not able to identify hematopoietic stem cells directly rather than being specific for hematopoietic stem cells.

[0006] To date, no cell surface marker for specifically identifying hematopoietic stem cells has been known.

[0007] Robo-4 is a cell surface receptor for Slit-2, and fetal and adult endothelial cells. Expressed in the cell. Highly expressed force in the heart Expression in the brain is very low. Robo-4 also inhibits migration of human microvascular endothelial cells (HMVEC).

[0008] Recently, Phillips et al. Reported a gene expression analysis at the genome level to elucidate the regulatory pathway of stem cells and its global genetic program. According to the report, Robo- 4 and Slit- 2 is, Lin- c- kit + Sca- l + Rho 1 ™ adult mouse bone marrow (BM) cells (Long hematopoietic stem cells: long- term HSCs). And Lin- AA4 1+ It was enriched in both c-kit + Sca- 1+ mouse fetal liver cells (fetal liver HSCs). Slit-2 is concentrated in both neural stem cells (NSC) and embryonic stem cells (ESC), and Robo-4 is a force concentrated in ESC. Slit-2 and Robo-4 are It was unconcentrated in HSCs. (Non-Patent Document 1)

However, Non-Patent Document 1 has no description or suggestion that Robo-4 is specifically expressed in hematopoietic stem cells.

[0010] Further, JP 2004-242513 describes a method for identifying and separating undifferentiated hematopoietic cells using JAM-1 as a marker (Patent Document 1). However, this document describes any markers specific to hematopoietic stem cells.

[0011] Therefore, development of a hematopoietic stem cell identification agent, a method for identifying hematopoietic stem cells, and a method for preparing a highly concentrated hematopoietic stem cell fraction has been awaited!

Patent Document 1: JP 2004-242513

Non-Patent Document 1: Phillips RL, et.al, Science. 288: 1635-40 (2000)

Disclosure of the invention

Problems to be solved by the invention

[0012] The present invention provides a hematopoietic stem cell identification agent, a method for identifying hematopoietic stem cells, and a method for preparing a highly concentrated fraction of hematopoietic stem cells.

Means for solving the problem

[0013] As a result of extensive research, the present inventor has unexpectedly found that Robo-4 is specifically expressed on the surface of hematopoietic stem cells, and has completed the present invention. . Therefore, the present invention relates to the following.

[0014] 1. Robo—Produced using 4 protein as an antigen and specifically binds to the protein A hematopoietic stem cell identification agent comprising an antibody.

2. The hematopoietic stem cell identification agent according to 1 above, wherein the antibody is a monoclonal antibody.

3. Antibody power The hematopoietic stem cell identification agent according to 2 above, which is a monoclonal antibody produced by Hypridoma whose accession number is ATCC PTA-6809, ATCC PTA-6810 or ATC C PTA-6811.

4. Robo—hematopoietic stem cell identification agent that also has 4 gene capabilities.

5. Robo—hematopoietic stem cell identification agent with 15 or more consecutive nucleotides that hybridizes under stringent conditions with the 4 gene.

6. A method for identifying hematopoietic stem cells using the hematopoietic stem cell identification agent according to any one of 1 to 5 above.

7. A method for preparing a highly enriched fraction of hematopoietic stem cells using the hematopoietic stem cell identification agent according to any one of 1 to 6 above.

The invention's effect

[0015] The present invention is useful for elucidating the mechanism of pluripotency in hematopoietic stem cell sorting, and for isolating and purifying hematopoietic stem cells for bone marrow transplantation and gene therapy.

Brief Description of Drawings

[0016] Fig. 1 shows the expression of Robo-4 mRNA in hematopoietic stem cells.

FIG. 2 shows the expression of Robo-4 mRNA in hematopoietic stem cells and hematopoietic progenitor cells.

FIG. 3 shows the expression of Robo-4 mRNA in the KSL-SP and KSL-MP fractions. BEST MODE FOR CARRYING OUT THE INVENTION

[0017] Robo-4 of the present invention is a single-membrane transmembrane protein having two integrin-like loop structures in the extracellular domain. Slit-2 is known as a ligand for Robo-4. Cells that express Robo4 move in the opposite direction to the concentration gradient of Slit2 by binding to Slit2. In the present invention, Robo-4 refers to a protein encoded by the gene having the base sequence shown in SEQ ID NO: 1 and having the amino acid sequence shown in SEQ ID NO: 2. For example, the mouse cDNA is GenBank accession number NM 028783, and the human cDNA is GenBank accession number NM 019055.

In the present invention, when identifying the Robo-4 protein of the present invention, the protein It is possible to use an antibody that is produced using the above polypeptide as an antigen and specifically binds to the polypeptide. Examples of the antibody include a monoclonal antibody and a polyclonal antibody. In addition, single chain antibodies produced by the phage display method or the like can also be used. The antibody can be prepared by a conventionally known method using the polypeptide of the present invention as an antigen. The antibody is preferably a monoclonal antibody.

Anti-Human Robo-4 monoclonal antibody can be used as Hypridoma ATCC? Ding Yachiichi 6809 (Clone 265721. 111), ATCC PTA- 6810 (Clone 265722. 111) or ATCC

PTA—6811 (clone 274914. 111) can be used to produce. These hybridomas were deposited internationally with the US ATCC, an international depositary organization, on June 21, 2000.

[0019] In order to identify hematopoietic stem cells in a test cell using the antibody of the present invention, an immunological assay using a known antibody can be used. Examples of the immunological measurement method include RIA method, ELISA method and fluorescent antibody method.

[0020] Using the agent for identifying hematopoietic stem cells of the present invention, the expression of Robo-4 gene and Z or Robo-4 protein in a test cell is identified, the identified hematopoietic stem cells are concentrated, and the hematopoietic stem cell highly enriched image Minutes can be prepared.

[0021] For example, in order to identify and concentrate hematopoietic stem cells using a fluorescent antibody method using the antibody of the present invention, the hematopoietic stem cells are labeled, and the labeled hematopoietic stem cells are separated by a cell sorter and collected. be able to.

For example, the antibody of the present invention can be fluorescently labeled and bound to an antigen expressed in hematopoietic stem cells to label the hematopoietic stem cells (direct fluorescent antibody method). Alternatively, hematopoietic stem cells can be labeled by binding an unlabeled antibody of the present invention to hematopoietic stem cells expressing an antigen and then binding a labeled secondary antibody (anti-immunoglobulin antibody) (intermediate). Fluorescent antibody method). The labeled hematopoietic stem cells can be identified and concentrated using a fluorescence activated cell sorter or the like.

[0023] Further, an unlabeled antibody of the present invention is contacted with hematopoietic stem cells, the antibody of the present invention is bound to an antigen expressed in the hematopoietic stem cells, and then contacted with a secondary antibody bound to magnetic beads. The Thus, the hematopoietic stem cells can be magnetically labeled.

[0024] Alternatively, the antibody of the present invention can be labeled with piotin, brought into contact with hematopoietic stem cells, bound to an antigen expressed in the hematopoietic stem cells, and then bound with streptavidin-labeled magnetic beads. Hematopoietic stem cells labeled with the magnetic beads can be concentrated using a magnetic separation column or the like.

[0025] In addition, in order to identify hematopoietic stem cells, a probe having a DNA sequence that hybridizes with the DNA sequence of the Robo-4 gene under stringent conditions can be used. Identification of hematopoietic stem cells using the probe can be appropriately performed using a known method. For example, DNA probes having an appropriate length, preferably 15 or more continuous, more preferably 20 or more consecutive nucleotides, are prepared from the DNA sequences shown in the sequence listing, and these probes are fluorescently labeled. Can be hybridized with a sample to identify hematopoietic stem cells.

[0026] As the DNA probe, it is possible to use a probe that also has all or part of the antisense strand of the base sequence of the gene of the present invention shown in the sequence listing.

[0027] The probe can also be used in the form of a microarray or DNA chip on which at least one or more are immobilized.

[0028] It should be noted that in the preparation of the DNA probe, in the base sequence of the present invention, conditions for "hybridization under stringent conditions" include, for example, hybridization at 42 ° C, and 1 X SSC (0. 15M NaCl, 0.015M sodium citrate), 0.1% SDS (Sodium dodecylsulfate) buffer solution at 42 ° C, and hybridization at 65 ° C. And a washing treatment at 65 ° C. with a buffer containing 0.1 × SSC and 0.1% SDS can be mentioned more preferably. There are various factors that affect the stringency of the hybridization, in addition to the above temperature conditions, and those skilled in the art can combine various elements to express the above stringency of the hybridization. It is possible to realize a stringency equivalent to that of a genie.

Example 1

[0029] Expression of Robo-1 to Robo-4 mRNA in hematopoietic stem cells Bone marrow mononuclear cells are collected from the mouse femur, and lineage marker-positive cells (fractional marker-positive cells) are removed using the MACS Lineage depletion kit (Miltenyl Biotec h), followed by anti-c-kit antibody and anti-Sea- Stained with 1 antibody. C-Kit + Sea-1 + Lineage (Lin) —cells (KSL) and Lineage + cells were sorted using FACS Vantage (Beckton Dickinson). RNA was extracted from these cells, and the amount of Robo4 mRNA was quantified by semi-quantitative RT-PCR. GAPDH, a housekeeping gene, was used as a control. As a result, only Robo-4 in the Robo family was found to express KSL specific expression.

Example 2

[0030] Expression of Robo-4 mRNA in hematopoietic stem cells and hematopoietic progenitor cells

High concentration of hematopoietic stem cells KSL cells, hematopoietic progenitor cells, myeloid common progenitor cells (CMP), megakaryocytes, erythroid progenitor cells (MEP), granulocyte Z macrophage progenitor cells (GMP), lymph Cellular common progenitor cells (CLP) have already been reported! FA CS sorting was performed by the method described above (Kondo M, et. Al. Cell 91: 661-672, 1997; Akashi K, et. Al. Natu re. 404 : 193-197, 2000). RNA was extracted from these cells, and the amount of Robo4 mRNA was measured by semi-quantitative RT-PCR. GAPDH was used as a control. As a result, Robo-4 expression was not observed in hematopoietic progenitor cells such as CMP, MEP, GMP, and CLP, and specific expression of Robo-4 was observed only with KSL.

Example 3

[0031] Expression of Robo-4 mRNA in KSL-SP and KSL-MP cells.

A group of cells that discharge Hoechst33342 is called a side population (SP). In addition, a main cell group constituting a cell group (non-SP) other than SP on FACS is called main population (MP). It is known that when KSL cells are further fractionated into SP and MP (KSL-SP, KSL-MP), hematopoietic stem cells are concentrated by the KSL-SP fraction. Mouse bone marrow Mononuclear cells were stained for KSL cells in the same manner as in Fig. 1, further stained with Hoechst33342, and KSL-SP cells, KSL-MP cells, KSL cells, and Lineage positive cells were collected by FACS. RNA was extracted from these cells, and the amount of Robo-4 mRNA was quantified by semiquantitative RT-PCR. GAPDH, a housekeeping gene, was used as a control. Robo-4 is most strongly expressed in the KSL-SP fraction containing the highest concentration of hematopoietic stem cells. In V, KSL—MP was strong!

Industrial applicability

The present invention is useful for elucidating the mechanism of pluripotency in hematopoietic stem cell sorting, and for isolating and purifying hematopoietic stem cells for bone marrow transplantation and gene therapy.

Claims

The scope of the claims

[1] Robo—A hematopoietic stem cell identification agent comprising an antibody that is produced using a protein 4 as an antigen and specifically binds to the protein.

[2] The hematopoietic stem cell identification agent according to claim 1, wherein the antibody is a monoclonal antibody.

[3] The antibody has an accession number of ATCC PTA-6809, ATCC PTA-6810 or ATCC

The hematopoietic stem cell identification agent according to claim 2, which is a monoclonal antibody produced by Hypridoma PTA 6811.

[4] Robo—hematopoietic stem cell identification agent that also has 4 gene power.

[5] Robo—A hematopoietic stem cell identification agent with 15 or more consecutive nucleotides that hybridizes with 4 genes under stringent conditions.

[6] A method for identifying hematopoietic stem cells using the hematopoietic stem cell identification agent according to any one of claims 1 to 5.

[7] A method for preparing a highly enriched fraction of hematopoietic stem cells using the hematopoietic stem cell identification agent according to any one of claims 1 to 6.