Patent Application
20080095746
g shows the results of subculturing moderately PCLP1-positive and weakly CD45, TER-119-positive or CD45, TER-119-positive cells with fresh OP9 cells.
h, 2-2i, and 2-2j show the culture obtained after co-culturing strongly PCLP1-positive and CD45, TER-119-negative or weakly CD45, TER-119-positive cells (fraction C) with OP9 cells for three, five, and seven days, respectively.
a shows the results of performing colony assays using a strongly PCLP1-positive fraction, moderately PCLP1-positive fraction, and weakly PCLP1-positive fraction.
b shows the results of assaying each fraction before co-culturing with OP9 cells.
c shows the results of performing colony assays using suspended cells produced by co-culturing each fraction with OP9 cells.
a and 8b show the endothelial cell-like colonies produced from PCLP1-positive cells derived from the spleen of an individual.
c and 8d show the blood cells produced from PCLP1-positive cells derived from the bone marrow of an individual.
a shows the results of assays using PCLP1-positive cells isolated from bone marrow.
b shows the results of assays using suspended cells produced as a result of co-culturing PCLP1-positive cells with OP9 cells.
a and 17c show the culture obtained after co-culturing bone marrow-derived CD34-positive, c-Kit-positive, PCLP1-positive cells with OP9 cells for ten and 15 days, respectively.
b and 17d show the culture obtained after co-culturing bone marrow-derived CD34-positive, c-Kit-positive, PCLP1-negative cells with OP9 cells for ten and 15 days, respectively.
a and 20b each show the results obtained by co-culturing the strongly PCLP1-positive fraction.
c, 20d, and 20e respectively show the results of culturing with the CD34-positive, c-Kit-positive, PCLP-negative fraction; the CD34-positive, c-Kit-positive, weakly PCLP1-positive fraction; and the CD34-positive, c-Kit-positive, strongly PCLP1-positive fraction.
The present invention is illustrated in detail below with reference to Examples.
14.5 days pregnant C57BL/6 mice
Phosphate buffered saline (PBS)
Liver perfusion medium (GIBCO BRL)
Collagenase/Dyspase solution (GIBCO BRL)
50 μg/mL gentamicin /15% fetal bovine serum (FBS)/DMEM (GIBCO
BRL)
2% FBS/PBS
OP9 cell line (Riken BioResource Center RCB 1124)
Anti-mouse CD16/32 monoclonal antibody (Pharmingen)
Biotinylated anti-mouse PCLP1 monoclonal antibody (MBL)
PE-labeled anti-mouse CD45 monoclonal antibody (Pharmingen)
PE-labeled anti-mouse TER-119 monoclonal antibody (Pharmingen)
7-AAD (Pharmingen)
Oncostatin M (OSM)
Basic fibroblast growth factor (bFGF)
Stem cell factor (SCF)
Various antibodies against mouse cell surface antigens
2% paraformaldehyde/PBS
Goat serum (Wako Pure Chemical Industries Ltd.)
Block Ace (Snow Brand Milk Products Co., Ltd.)
MethoCult (StemCell Technologies)
Pregnant mice were euthanized by cervical dislocation, and the uterus was removed. The uterine wall was then removed in PBS, and the fetuses were extirpated. After changing to fresh PBS, the fetal livers were extirpated under a stereoscopic microscope, and the medium was exchanged to 12 mL of liver perfusion medium per litter of fetuses (six to 12 fetuses). All of the following procedures were performed under sterile conditions.
The fetal livers were cut into small pieces using a pair of surgical scissors, and the medium was exchanged to 12 mL of Collagenase/Dyspase solution per litter of fetuses (six to 12 fetuses). This was incubated in a CO2 incubator at 37° C. for ten minutes, and then subjected to enzyme treatment. The tissue structure was destroyed by thorough pipetting using a 10-mL glass pipette to suspend the cells. This was transferred to a centrifuge tube, an equivalent amount of 50 μg/mL gentamicin/15% FBS/DMEM was added and mixed, and this was centrifuged at 4° C. and 800 rpm for ten minutes. The supernatant was removed, and 15 mL of an ice-cooled hemolysis buffer (0.1 M NH4Cl/16.5 mM Tris) was added per litter of fetuses (six to 12 fetuses), the cells were loosened by gently pipetting two to three times, and this was left on ice for nine minutes for hemolysis. An equivalent amount of 50 μg/mL gentamicin/15% FBS/DMEM was added, and this was centrifuged at 4° C. and 800 rpm for ten minutes. The collected cells were diluted in 10 mL of 50 μg/mL gentamicin/15% FBS/DMEM, and this was passed through a 70 μum cell strainer. Dead cells were stained with trypan blue, and the number of stained cells was counted using a hemocytometer.
Anti-mouse CD16/32 monoclonal antibody was diluted 100 times with 50 μg/mL gentamicin/15% FBS/DMEM. 1 mL of this solution was added per 1×107 cells, this was mixed, then left on ice for 15 minutes, and non-specific binding of antibodies was inhibited by FcR blocking. About 1×106 cells were placed into each of three tubes, and the antibodies below were added to the respective tubes and then mixed to produce isotype controls and samples for fluorescence correction. Each of the antibodies was added such that they were diluted 100 times.
Tube 1: biotinylated rat IgG2a and PE-labeled rat IgG2a
Tube 2: biotinylated anti-mouse CD45 monoclonal antibody and PE-labeled rat IgG2a
Tube 3: biotinylated rat IgG2a and PE-labeled anti-mouse CD45 monoclonal antibody
Biotinylated anti-mouse PCLP1 monoclonal antibody, PE-labeled anti-mouse CD45 monoclonal antibody, and PE-labeled anti-mouse TER-119 monoclonal antibody were added to the remaining cells such that they were each diluted 100 times, and this was mixed to prepare the samples. After adding the antibodies, the respective cells were left on ice for 30 minutes.
The isotype controls, fluorescence correction samples, and samples were each washed with ice-cooled 2% FBS/PBS. The isotype controls, fluorescence correction samples, and samples were then each rediluted in streptavidin-APC diluted 50 times with 2% FBS/PBS, and then left on ice for 30 minutes. They were then washed with ice-cooled 2% FBS/PBS. Cells were diluted at 1×106 cells per 5 μL of 7-AAD, and then left at room temperature for five minutes. The cells were diluted in 2% FBS/PBS or diluted in PBS to 5×106 to 1×107 cells/mL, and then transferred to a cell separator tube.
Using isotype controls and fluorescence correction samples, the sensitivity of each parameter of the cell separator was adjusted and fluorescence corrections were made. With reference to the fluorescence intensity of the isotype control, each of the cell populations below were gated, and the cells were fractionated into tubes containing 50 μg/mL gentamicin/15% FBS/DMEM mixed with 10 μg/mL OSM, 1 μg/mL bFGF, and 100 μg/mL SCF.
Strongly PCLP1-positive and CD45, TER-p119-negative or weakly CD45, TER-19-positive Moderately PCLP1 positive and weakly CD45, TER-119-positive or CD45, TER-119-positive PCLP1-negative and strongly CD45, TER-119-positive
The fractionated cells were reanalyzed to confirm that they were purely fractionated according to the gates that were set. The number of obtained cells was counted using a hemocytometer.
On a 10-cm dish or 6-well plate, OP9 stromal cells were cultured in 50 μg/mL gentamicin/15% FBS/DMEM until about 70% to 90% confluent. Immediately before plating the sorted cells onto this dish or plate, the medium was exchanged for a medium containing cytokines (10 μg/mL OSM, 1 μg/mL bFGF, and 100 μg/mL SCF). The strongly PCLP1-positive and CD45, TER-119-negative or weakly CD45, TER-119-positive fraction were plated onto a 6-well plate at about several hundred to 5000 cells per well, and the moderately PCLP1-positive and weakly CD45, TER-119-positive or CD45, TER-119-positive fraction and the PCLP1-negative and strongly CD45, TER-119-positive fraction were each plated at 20 000 cells per well. The cells were cultured in a CO2 incubator at 37° C. under 5% CO2 partial pressure. Blood cell production in each of the fractions was observed under a microscope for several weeks, starting the day after plating.
The sorted cells were added to MethoCult to a concentration of 1000 cells/mL, to which a final concentration of 50 μg/mL gentamicin was added and mixed. 1 mL of this solution was placed into each well of a 6-well plate using a 1-mL syringe and an 18 G needle. To retain moisture, 1 mL of sterilized distilled water or PBS was placed onto a corner of the plate, and the cells were cultured in a CO2 incubator at 37° C. under 5% CO2 partial pressure. On Day 9 of culturing, the colonies were observed under a microscope, the colony types were classified, and then counted.
After several days of OP9 co-culture, and after producing a sufficient amount of suspended cells, the suspended cells were exclusively collected into a centrifuge tube, taking care to avoid OP9 contamination. The obtained cells were used to analyze cell surface antigen expression using flow cytometry, and to measure growth activity of the blood cells using colony assays.
The dish was washed with PBS, taking care not to detach the cells. The cells were immobilized with 2% paraformaldehyde/PBS and then 5% goat serum/BlockAce (Snow Brand Milk Products Co., Ltd.) was used for blocking (for one hour at room temperature). A primary antibody reaction was carried out overnight at 4° C., and then washing was carried out using PBS. A fluorescence-labeled anti-mouse IgG antibody (secondary antibody) reaction was carried out for one hour at room temperature, and after washing with PBS, the cells were observed under a microscope.
The results showed that the expression of PCLP1 in E14.5 fetuses can be classified into four groups according to the intensity of expression: highly positive (approximately 1%), moderately positive (approximately 40%), weakly positive (approximately 40%), and negative (approximately 15%) (
Observation of each OP9 co-cultured fraction under a phase-contrast microscope showed that on Day 2 to 3 of culture many cobble-stone area-forming cells (CAFCs), which appear black because they are under OP9 stromal cells, were observed in the wells plated with PCLP1-negative and strongly CD45, TER-119-positive cells, and formation of many white glowing suspended cells around these CAFCs was observed (
In the wells plated with moderately PCLP1-positive and weakly CD45, TER-119-positive or CD45, TER-119-positive cells, the white glowing suspended cells started to grow around the CAFCs from approximately Day 7 to 10 of culture (
On the other hand, at about Day 3 to 6 of culturing in wells plated with strongly PCLP1-positive and CD45, TER-119-negative or weakly CD45, TER-119-positive cells, endothelial-like colonies formed and grew at a rate of approximately 10% of the number of plated cells (
The endothelial-like colonies produced by co-culturing the strongly PCLP1-positive fraction with OP9 were immunohistologically stained using various types of endothelial cell surface antigens. As a result, CD34, CD31, and VE-Cadherin were clearly stained as compared to the isotype control (
Suspended cells, derived from the moderately PCLP1-positive and weakly CD45, TER-119-positive or CD45, TER-119-positive fractions produced by OP9 co-culture, were collected on Day 10 of culture, and when the expression of cell surface antigens was analyzed by flow cytometry, nearly 100% of the cells expressed leukocyte cell surface antigen CD45, and cell surface antigens of hematopoietic stem cells and hematopoietic precursor cells, such as CD34, c-Kit, Sca-1, and CD31, were highly expressed (
In the colony assays, the Colony Forming Units (CFU) were taken as the number of colonies formed for every 10 000 cells plated, and CFU-C indicates the total number of colonies formed. CFU- followed by a letter of the alphabet indicates the number of colonies formed by each type of differentiated blood cell, and G, M, Meg, E, and Mix respectively refer to granulocytes, monocytes and niacrophages, megakaryocytes, erythroblasts, and a mixture of all types of cells. Blood cell colony formation was hardly observed from the strongly PCLP1-positive, moderately PCLP1-positive, and weakly PCLP1-positive fractions. The number of colonies formed from the PCLP1-negative fraction was CFU−C=670 per 10 000 cells, and that in the entire liver was CFU−C=63.3 per 10 000 cells (
Colony assays using suspended cells produced by OP9 co-culture showed that the number of colonies formed from suspended cells derived from the moderately PCLP1 positive and weakly CD45, TER-119-positive or CD45, TER-119-positive fraction and the PCLP1-negative and strongly CD45-, TER-119-positive fraction were CFU-C=1276.7 and CFU-C =543.3, respectively, and the colony forming ability of both types of cells increased remarkably compared to that observed prior to OP9 co-culture (
Immunostaining of the endothelial-like colonies produced by OP9 co-culture revealed the expression of CD34 and VE-Cadherin, which are endothelial cell surface antigens. The endothelial-like colonies together with OP9 were trypsinized, then the cells were dispersed and replated on to fresh OP9, once again causing formation and growth of endothelial-like colonies.
The fraction of PCLP1-negative and strongly CD45, TER-119-positive cells is a cell population comprising blood cell precursor cells that can immediately provide functional blood cells; therefore, this fraction was thought to begin active blood cell growth from the early stages of culture. In contrast, the fractions of moderately PCLP1-positive and weakly CD45, TER-119-positive or moderately CD45, TER-119-positive cells comprise juvenile blood cells that are still in the process of differentiation, and thus it is some time before blood cell growth starts when these cells are co-cultured with OP9 stromal cells. Accordingly, these fractions are thought to begin blood cell precursor cell production later compared to the PCLP1-negative and strongly CD45, TER-119-positive cell fraction, and this blood cell growth continued for a long time.
Since suspended cells derived from moderately PCLP1-positive cells were the only cells that could be subcultured, and they could sustain longer-term production of blood cells, this fraction is likely to contain blood cell stem cells that can self-replicate. Further, since both the PCLP1-negative and strongly CD45, TER-119-positive cell fraction and the moderately PCLP1 positive and weakly CD45, TER-119-positive or CD45, TER-119-positive cell fractions showed remarkably different colony forming abilities before and after OP9 co-culture, and since both showed a considerable increase in colony forming ability after OP9 co-culture, OP9 co-culturing appears to strongly induce blood cell differentiation and growth.
The results of cell surface antigen expression analysis by flow cytometry were that the strongly PCLP1-positive cells were negative or weakly positive for known endothelial cell surface antigens CD34, CD31, and Flk-1. However, when these fractions were OP9 co-cultured, they frequently formed endothelial-like colonies that were positive for the endothelial cell surface antigens CD34 and VE-Cadherin. Therefore, it was not until after co-culturing with stromal cells that the strongly PCLP1-positive cell fraction differentiated into endothelial cells, and these fractions may be cell populations comprising endothelial precursor cells that may acquire the properties of endothelial cells. This indicates that using anti-PCLP1-antibodies enables the separation of more juvenile endothelial precursor cells not obtainable using existing cell surface antigens.
The above showed that by combining the level of PCLP1 expression with information on CD45 and TER-119 expression, a cell fraction comprising blood cell precursor cells, a cell fraction comprising more juvenile blood cell stem cells, and a cell fraction comprising endothelial precursor cells can each be separated. It also showed that co-culturing with OP9 stromal cells can strongly induce in vitro blood cell differentiation and growth, and that this growth activity can be maintained for a long time.
Newborn C57BL6 mice
PBS
Collagenase/Dyspase solution (GIBCO BRL)
50 μg/mL gentamicin/15% FBS/DMEM (GIBCO BRL)
2% FBF/PBS, OP9 cell line
Anti-mouse CD16/32 monoclonal antibody (Pharmingen)
Biotinylated anti-mouse PCLP1 monoclonal antibody (MBL)
APC-labeled anti-mouse c-Kit monoclonal antibody (Pharmingen)
FITC-labeled anti-mouse CD34 monoclonal antibody (Pharmingen)
Streptavidin-APC (Molecular Probes)
7-AAD (Pharmingen), Oncostatin M (OSM)
Basic fibroblast growth factor (bFGF)
Stem cell factor (SCF)
Various antibodies against mouse cell surface antigens
MethoCult (Stem Cell Technologies)
The spleen and bone marrow were extirpated from newborn mice. The spleen or bone marrow from a litter of fetuses (six to 12 fetuses) was soaked in 12 mL of Collagenase/Dyspase solution, cut into small pieces using a pair of surgical scissors, incubated at 37° C. for ten minutes in a CO2 incubator, and then subjected to enzyme treatment. After thorough pipetting using a 10-mL glass pipette, the cells were dispersed, transferred to a centrifuge tube, an equivalent amount of 50 μg/mL gentamicin/15% FBS/DMEM was added and mixed, and this was centrifuged at 4° C. and 800 rpm for ten minutes. The supernatant was removed, the cells were resuspended in 50 μg/mL gentamicin/15% FBS/DMEM, and the number of cells was counted using a hemocytometer.
Anti-mouse CD 16/32 monoclonal antibody was diluted 100 times with 50 μg/mL gentamicin/15% FBS/DMEM, and 0.1 mL of this solution was added per 1×106 of spleen or bone marrow cells. This was then mixed, the mixture was left on ice for 15 minutes, and non-specific antibody binding was inhibited by FcR blocking. About 1×105 cells were placed into each of four tubes, and the antibodies below were added to the respective tubes. This was then mixed to produce isotype controls and samples for fluorescence correction. Each of the antibodies was added such that they were diluted 100 times.
Tube 1: FITC-labeled rat IgG2a, PE-labeled rat IgG2a, and biotinylated rat IgG2a
Tube 2: FITC-labeled anti-mouse CD45 monoclonal antibody, PE-labeled rat IgG2a, and biotinylated rat IgG2a
Tube 3: FITC-labeled rat IgG2a, PE-labeled anti-mouse CD45 monoclonal antibody, and biotinylated rat IgG2a
Tube 4: FITC-labeled anti-rat IgG2a, PE-labeled rat IgG2a, and biotinylated anti-mouse CD45 monoclonal antibody
Biotinylated anti-mouse PCLP1 monoclonal antibody, APC-labeled anti-mouse c-Kit monoclonal antibody, and FITC-labeled anti-mouse CD34 monoclonal antibody were added to the remaining cells such that they were then each diluted 100 times, and this was mixed to prepare the samples. After adding the antibodies, the respective cells were left on ice for 30 minutes. The isotype controls, fluorescence correction samples, and the samples were each washed with ice-cooled 2% FBS/PBS. The isotype controls, fluorescence correction samples, and samples were each rediluted in streptavidin-APC diluted 50 times with 2% FBS/PBS, and then left in ice for 30 minutes. They were then washed with ice-cooled 2% FBS/PBS. Cells were diluted at 1×106 cells per 5 μL 7-AAD, and then left at room temperature for five minutes. The cells were diluted in 2% FBS/PBS or diluted in PBS to a concentration of 2×106 to 5×106 cells/mL, and then transferred to cell separator tubes.
Using isotype controls and fluorescence correction samples, the sensitivity of each parameter of the cell separator (cell sorter) was adjusted and fluorescence corrections were made. The samples were developed according to the fluorescence intensity of PCLP1 and cell size (FS peak). With reference to the fluorescence intensity of the isotype control, the strongly PCLP1-positive, moderately PCLP1-positive, weakly PCLP1-positive, and PCLP1-negative regions in the sample were each gated, and in the combined staining for CD34 and c-Kit, the relationship between each cell surface antigen was analyzed and gates were set for these surface antigens. The groups of sorted cells were fractionated in tubes containing 50 μg/mL gentamicin/15% FBS/DMEM, and the number of cells were counted using a hemocytometer.
On a 10-cm dish or 6-well plate, OP9 stromal cells were cultured in 50 μg/mL gentamicin/15% FBS/DMEM until about 70% to 90% confluent, and then a suitable number of sorted cells were plated to this culture after replacing the medium with a medium containing cytokines (10 μg/ml OSM, 1 μg/ml bFGF, 100 μg/ml SCF). The co-culture dish was incubated in a CO2 incubator at 37° C. under 5% CO2 partial pressure. Blood cell production in each of the fractions was observed under a microscope for several weeks, starting the day after plating.
The results showed that expression of PCLP1 in the spleen can be categorized into four groups, according to expression intensity: strongly positive (PCLP1++; approximately 1%), moderately positive (PCLP1+; approximately 30%), weakly positive (PCLP1low; approximately 18%), and negative (PCLP1−; approximately 51%) (
By Day 10 of culture the strongly PCLP1-positive fraction was found to be forming many endothelial cell-like colonies, morphologically similar to the endothelial cell-like colonies formed from an OP9 co-culture of strongly PCLP1-positive fetal liver cells (
The results showed that the expression of PCLP1 in bone marrow could be categorized into four groups, according to expression intensity: strongly positive (approximately 1%), moderately positive (approximately 14%), weakly positive (approximately 8%), and negative (approximately 77%) (
When PCLP1-positive cells were co-cultured with OP9 stromal cells, the suspended blood cells were observed to form in clusters within one week of culturing, and cobble stone-like cells could also be recognized. On Day 11 of culturing, the suspended cells grew vigorously, appearing as a sea of clouds, and OP9 cells could not be observed at all (
PCLP1-positive cells were separated from the bone marrow and then subjected to colony assays, showing activity of CFU-G=2.2, CFU-M=75.6, CFU-GM=5.6, CFU-E=33.3, CFU-Mix=1.1, and CFU-C=117.8 per 10000 cells (
The results showed that strongly PCLP1-positive cells exist in the tissues of individuals, although in low frequency, and OP9 co-culturing produces endothelial-like colonies that are morphologically similar to those produced from the strongly PCLP1-positive cells of fetal liver. Furthermore, as for fetal liver, blood cell growth in PCLP1-positive cells was activated later than in PCLP1-negative cells. The above results showed that during the developmental process of a fetus, as the site of hematopoiesis shifts from the AGM region, where adult-type hematopoiesis begins, to the liver and tissues of an individual, a strongly PCLP1-positive cell fraction and a moderately PCLP1-positive cell fraction are consistently present in each of the hematopoietic organs, and throughout the transition, the strongly PCLP1-positive cell fraction consistently includes a high frequency of endothelial precursor cells, and the moderately positive cells include hematopoietic stem cell-like juvenile cells that continuously produce blood cells for a long time.
The results also demonstrated that the use of a stromal cell co-culturing system, such as the OP9 co-culturing system, enables ex vivo or in vitro growth of immature precursor cells derived from the tissues of individuals over a long time. CD34-positive and c-Kit-positive cell fractions are cell fraction with hematopoietic stem cells and hematopoietic precursor cell fractions concentrated to a certain degree, but the PCLP1-positive cell population within this fraction is very probably a subfraction which is at a different stage of blood cell differentiation. That is, within the hematopoietic stem cell and hematopoietic precursor cell fractions, the cell populations expressing PCLP1 may be more juvenile. However, the colony forming ability of suspended cells after co-culturing with stromal cells tended to be higher for cells derived from PCLP1-negative fractions. Since only the PCLP1-positive fractions continued to produce blood cells for several weeks thereafter, at this point the PCLP1-positive fractions may not have reached the stage of blood cell differentiation with the highest growth activity.
Human bone marrow monocytes (BMMC) were purchased as frozen cells from Cambrex (Japanese supplier: Sanko Junyaku Co., Ltd.) and then used. The CHO cells used for gene transfer were purchased from the Riken BioResource Center and then subcultured in F12HAM medium (SIGMA) containing 10% FBS (MBL) and 50 μg/mL gentamicin (GIBCO).
Human PCLP1 cDNA was cloned from a human placenta library, and the full length sequence and extramembrane region sequence were used to make constructs for expression in animal cells using the pcDNA3.1 vector (Invitrogen). The structure of the constructs is shown in
The secretory PCLP1-expressing cell line 18E was cultured for one week in 1 L of 10% FBS-F12HAM medium, the culture was dialyzed overnight at 4° C. against PBS, and then purified using a WGA-Sepharose column (Amersham). Recombinant PCLP1 that adhered to the column was eluted with PBS containing 200 mM N-acetylglucosamine, the eluted fractions were dialyzed again against a phosphate buffer solution (pH 7.4), and then adsorbed onto DEAE-Sepharose (Amersham). The recombinant adsorbed onto the column was eluted with PBS containing 1 M NaCl, then the eluted fractions were combined, diluted 5-fold using a phosphate buffer (pH 7.4), and then adsorbed onto a ConA Sepharose (Amersham). The recombinant adsorbed onto the column was eluted using PBS with a concentration gradient of 0-200 mM α-dimethylglucose, and the fractions reactive to myc tag antibody (MBL) were collected as purified products (
Western blotting was carried out to confirm that the transgenic cells and the purified protein are the desired recombinant PCLP1 protein. After subjecting the sample to electrophoresis on a 10% polyacrylamide gel, the proteins were electrically transferred from the gel onto a PVDF membrane (Millipore). The transferred membrane was blocked overnight in 5% skim milk-PBS at 4° C. The membrane was washed with PBS, then reacted with a 2000-fold diluted anti-myc tag antibody (MBL) at room temperature for one hour. After washing with PBS, this was reacted with a 3000-fold diluted peroxidase-labeled anti-mouse IgG (H+L ) antibody (MBL) at room temperature for one hour. The membrane was washed thoroughly with PBS, color developed with SuperSignal coloring substrate, and an X-ray film (Fuji Film) was then exposed using the resultant signals.
100 μL of complete adjuvant (Iatron) was pre-injected to Balb/c mice, and one day later, the transgenic cells suspended in PBS were used to immunize the mice four times at three-day intervals, using 1×106 cells for each immunization. Two days after the final immunization, the lymph nodes were extirpated from the mice, a P3U1 myeloma cell line was added at ⅓ the equivalent of the total number of lymphocytes, and cell fusion was carried out using polyethylene glycol (WAKO). The fused cells were cultured for two weeks in HAT medium (GIBCO) to select only the fused cells. Flow cytometry was used to confirm the reactivity of culture supernatant of the obtained fused cells (hybridomas) toward the transgenic cells, and highly reactive hybridomas were subcultured. The hybridomas were cultured in 1 L of 10% FBS-RPMI medium, the culture supernatant was dialyzed against PBS, and then adsorbed onto a Protein A column (Amersham). The adsorbed monoclonal antibodies were eluted with 0.17 M glycine-HCl buffer (pH 4.0), and the eluted fractions were combined and dialyzed against PBS. Some of the monoclonal antibodies were biotinylated using EZ-Link Biotinylation Kit (PIERCE) with the aim of confirming their flow cytometry reactivity.
The monoclonal antibodies used for flow cytometry and cell sorting included CD45-PE, CD45-FITC, CD117-PE, CD34-PE, IgG2a-FITC, IgG1-FITC, IgG2a-PE, IgG1-PE, and streptavidin FITC; all were products from Immunotech. The frozen cells were thawed at 37° C., washed with IMDM medium (SIGMA) containing 10% FBS (MBL), and then suspended in 5% FBS-PBS. 50 μg/mL of biotin-labeled PCLP1 antibody and commercially available PE-labeled antibody were simultaneously reacted in ice for one hour. The cells were washed several times in 5% FBS-PBS, and then reacted with streptavidin-FITC for 20 minutes in ice. The cells were washed and then suspended in 5% FBS-PBS to a concentration of 5×106 cells/mL. Analysis and cell fractionation were carried out using a Beckman Coulter Epics Altra.
Full-length and extramembrane region PCLP1 genes were forcibly expressed in CHO cells, and cell lines each stably expressing the recombinant proteins were established (
Hybridomas (clone 53D11 and such) that produce anti-human PCLP1 monoclonal antibodies were established by immunizing mice with the gene expression cell line. Anti-human PCLP1 monoclonal antibodies were purified from the hybridoma culture supernatant to produce biotinylated antibodies and the like. The obtained antibodies were confirmed to have reactivity against the cell line (12C) expressing the full-length human PCLP1 protein (
The produced monoclonal antibodies were confirmed to have reactivity against bone marrow (
Monoclonal antibodies that recognize the human PCLP1 molecule were produced using materials in which recombinant proteins are expressed in animal cells. The percentage of PCLP1 molecule-expressing cells in the human bone marrow is very low, less than 1%, and the distribution of these cells hardly overlaps with the distribution of the hematopoietic stem cell (CD34) population. This finding matches the expression pattern in murine bone marrow.
C57BL6 mice
PBS
Collagenase/Dyspase solution (GIBCO BRL)
50 μg/mL gentamicin/15% FBS/DMEM (GIBCO BRL)
2% FBS/PBS, OP9 cell line
Anti-mouse CD16/32 monoclonal antibody (Pharmingen)
Biotinylated anti-mouse PCLP1 monoclonal antibody (MBL)
APC-labeled anti-mouse c-Kit monoclonal antibody (Pharmingen)
FITC-labeled anti-mouse CD34 monoclonal antibody (Pharmingen)
Streptavidin-APC (Molecular Probes)
7-AAD (Pharmingen)
Oncostatin M (OSM), Basic fibroblast growth factor (bFGF)
Stem cell factor (SCF)
Various antibodies against mouse cell surface antigens
MethoCult (Stem Cell Technologies)
The mice were euthanized by being left in ice for ten minutes. Their femurs were extirpated under a stereoscopic microscope. The femurs of six to twelve individuals were soaked in 12 mL of Collagenase/Dyspase solution, and then broken into pieces using a pair of tweezers. This was incubated in a CO2 incubator at 37° C. for ten minutes, and the whole, including the bones, was subjected to enzyme treatment. The cells were suspended by thorough pipetting using a 10 mL glass pipette, and then filtered for transfer into a centrifuge tube. An equivalent amount of 50 μg/mL gentamicin/15% FBS/DMEM was added and mixed in, and this was centrifuged at 4° C. and 1200 rpm for ten minutes. The supernatant was removed, the residue was resuspended in 50 μg/mL gentamicin/15% FBS/DMEM, and the number of cells was counted using a hemocytometer.
Anti-mouse CD16/32 monoclonal antibody was diluted 100 times with 50 μg/mL gentamicin/15% FBS/DMEM, and 0.1 mL of this solution was added to every 1×106 bone marrow cells and mixed. The mixture was left on ice for 15 minutes, and non-specific antibody binding was inhibited by FcR blocking. About 1×105 cells were placed into each of four tubes, and the antibodies below were added to the respective tubes and mixed to produce isotype controls and samples for fluorescence correction. Each of the antibodies was added such that they were diluted 100 times.
Tube 1: FITC-labeled rat IgG2a, PE-labeled rat IgG2a, and biotinylated rat IgG2a
Tube 2: FITC-labeled anti-mouse CD45 monoclonal antibody, PE-labeled rat IgG2a, and biotinylated rat IgG2a
Tube 3: FITC-labeled rat IgG2a, PE-labeled anti-mouse CD45 monoclonal antibody, and biotinylated rat IgG2a
Tube 4: FITC-labeled anti-rat IgG2a, PE-labeled rat IgG2a, and biotinylated anti-mouse CD45 monoclonal antibody
Biotinylated anti-mouse PCLP1 monoclonal antibody, APC-labeled anti-mouse c-Kit monoclonal antibody, and FITC-labeled anti-mouse CD34 monoclonal antibody were added to the remaining cells such that they were each diluted 100 times, and this was then mixed to prepare the samples. These were left on ice for 30 minutes, and then the isotype controls, fluorescence correction samples, and samples were each washed with ice-cooled 2% FBS/PBS. The isotype controls, fluorescence correction samples, and samples were each rediluted in streptavidin-APC diluted 50 times with 2% FBS/PBS, and then left in ice for 30 minutes. They were then washed with ice-cooled 2% FBS/PBS. Cells were diluted at 1×106 cells per 5 μL 7-AAD, and then left at room temperature for five minutes. The cells were diluted in 2% FBS/PBS or diluted in PBS to a concentration of 2×106 to 5×106 cells/mL, and then transferred to a cell separator tube.
Using isotype controls and fluorescence correction samples, the sensitivity of each parameter of the cell separator (cell sorter) was adjusted and fluorescence corrections were made. With reference to the fluorescence intensity of the isotype control, the CD34-positive, c-Kit-positive cell population in the sample was gated, and gates were further developed within these gates according to PCLP1 expression levels; two sorting gates were set, one for CD34-positive, c-Kit-positive, and PCLP1-positive cells; and another for CD34-positive, c-Kit-positive, and PCLP1-negative cells. The sorting gates were set such that when the CD34-positive, c-Kit-positive cell fraction is defined as 100%, the PCLP1-negative subfraction will be approximately 58% and the PCLP1-positive subfraction will be approximately 15%. The cells were fractionated in tubes containing 50 μg/mL gentamicin/15% FBS/DMEM. The fractionated cells were reanalyzed to confirm that they were purely fractioned according to the gates that were set. After fractionation the cells were centrifuged and the supernatant removed to reduce the volume of the solution, as necessary. The number of obtained cells was counted using a hemocytometer.
On a 10-cm dish or 6-well plate, OP9 stromal cells were cultured in 50 μg/mL gentamicin/15% FBS/DMEM until approximately 70% to 90% confluent. Immediately before plating the sorted cells onto this dish or plate, the medium was replaced with a medium containing cytokines (10 μg/mL OSM, 1 μg/mL bFGF, and 100 μg/mL SCF). The CD34-positive, c-Kit-positive, and PCLP1-positive fraction, and the CD34-positive, c-Kit-positive, and PCLP1-negative fraction obtained by sorting were each plated into 6-well plates at 3000 cells per well. The cells were cultured in a CO2 incubator at 37° C. under 5% CO2 partial pressure conditions, and blood cell production in each of the fractions was observed under a microscope for several weeks, starting from the day after plating.
After several days of OP9 co-culture, and after producing a sufficient amount of suspended cells, the suspended cells were exclusively collected into centrifuge tubes, taking care to avoid OP9 contamination. The obtained cells were used to analyze the expression of cell surface antigens using flow cytometry, and to measure growth activity of the blood cells using colony assays.
Expression of PCLP1 in the bone marrow could be categorized into four groups according to expression intensity: strongly positive (approximately 1%), moderately positive (approximately 14%), weakly positive (approximately 8%), and negative (approximately 77%) (
Observation of each fraction under a phase contrast microscope after OP9 co-culture showed that during Days 7 to 14 of culture, the CD34-positive, c-Kit-positive, and PCLP1-positive fraction formed blood cell clusters at a few places in the dish, with growth of suspended blood cell-like cells observed to a degree. In contrast, the CD34-positive, c-Kit-positive, and PCLP1-negative fraction produced such a large amount of suspended blood cell-like cells that the OP9 stromal cells could not be observed (
However, by the beginning of the third week of culture, the blood cell growth activity in these two fractions was reversed, and blood cell production gradually ceased in the PCLP1-negative fraction, while blood cell production gradually became more active in the PCLP1-positive fraction (
When suspended cells of both fractions were each collected on Day 15 of OP9 co-culture, a sufficient amount of cells could not be obtained from the PCLP1-negative fraction, therefore, analysis of cell surface antigen expression by flow cytometry was only carried out for the PCLP1-positive fraction. The results showed that nearly 100% of cells expressed CD45, and that cell surface antigens of hematopoietic stem cells and hematopoietic precursor cells, such as CD34, c-Kit, and CD31, were expressed very frequently (
When suspended cells of both fractions were each collected on Day 15 of OP9 co-culture, colony assays were performed, and the results were that CFU-C=753.3 per 10 000 cells regarding the suspended cells derived from the PCLP1-positive fraction, and CFU-C=1583.3 per 10 000 cells regarding the suspended cells derived from the PCLP1-negative fraction (
The use of the OP9 co-culture system indicated that bone marrow cells can also grow for a long time ex vivo or in vitro. The CD34-positive, c-Kit-positive cell fraction are thought to be a fraction with hematopoietic stem cells and hematopoietic precursor cell fractions concentrated to some degree; however, when this fraction was further fractioned into PCLP1-positive and PCLP1-negative subfractions, the time required for blood cell growth to start was significantly different for each of the fractions, and therefore the subfractions are highly likely to be at different differentiation stages of blood cell differentiation.
That is, in hematopoietic stem cell and hematopoietic precursor cell fractions, the low frequency cell populations expressing PCLP1 may be more juvenile. However, the colony forming ability of suspended cells on Day 15 of culture was twice as much or more for cells derived from the PCLP1-negative fraction than for the PCLP1-positive fraction. Since only the PCLP1-positive fraction continued to produce blood cells for several weeks thereafter, it is thought that the above results were because at this point the PCLP1-positive fraction may not have reached the differentiation stage of blood cell differentiation with highest growth activity.
In the AGM region, where hematopoiesis is said to begin during embryonic development, approximately 90% of CD34-positive cells express PCLP1 (WO 01/34797). The present results further confirmed that as the site of hematopoiesis gradually shifts from the AGM to the fetal liver and bone marrow, the proportion of PCLP1-positive cells among the CD34-positive cells dramatically decreases from 50% or so in the fetal liver to a few percent or so in the bone marrow (
Experimental results supporting this theory include the phenomenon that in a system of co-culture with stromal cells, in terms of the time taken until hematopoietic activity is observed, the CD34-positive, c-Kit-positive, PCLP1-negative cells start to show blood cell growth at a relatively early stage and complete the growth in a short time, whereas, the CD34-positive, c-Kit-positive, PCLP1-positive cell population takes a long time until blood cell growth starts and continues to produce blood cells for a long time (
Newborn C57BL/6 mice
PBS
Collagenase/Dyspase solution (GIBCO BRL)
50 μg/mL gentamicin/15% FBS/DMEM (GIBCO BRL)
2% FBS/PBS
OP9 cell line
Anti-mouse CD16/32 monoclonal antibody (Pharmingen)
Biotinylated anti-mouse PCLP1 monoclonal antibody (MBL)
APC-labeled anti-mouse c-Kit monoclonal antibody (Pharmingen)
FITC-labeled anti-mouse CD34 monoclonal antibody (Pharmingen)
Streptavidin-APC (Molecular Probes)
7-AAD (Pharmingen)
Oncostatin M (OSM)
Basic fibroblast growth factor (bFGF)
Stem cell factor (SCF)
Various antibodies against mouse cell surface antigens
MethoCult (Stem Cell Technologies)
The newborn mice were euthanized by being left in ice for ten minutes. Their spleen was extirpated under a stereoscopic microscope. The spleens were soaked in Collagenase/Dyspase solution at 12 mL of Collagenase/Dyspase solution for a litter of fetuses (six to twelve fetuses), and then cut into small pieces using a pair of surgical scissors. This was incubated in a CO2 incubator at 37° C. for ten minutes, and then subjected to enzyme treatment.
Cells were dispersed by thorough pipetting using a 10-mL glass pipette. These cells were transferred to a centrifuge tube, an equivalent amount of 50 μg/mL gentamicin/15% FBS/DMEM was added and mixed, and this was centrifuged at 4° C. and 800 rpm for ten minutes. The supernatant was removed, the residue was resuspended in 50 μg/mL gentamicin/15% FBS/DMEM, and the number of cells was counted using a hemocytometer.
Anti-mouse CD16/32 monoclonal antibody was diluted 100 times with 50 μg/mL gentamicin/15% FBS/DMEM, and 0.1 mL of this solution was added per 1×106 spleen cells. This was then mixed, the mixture was left on ice for 15 minutes, and non-specific antibody binding was inhibited by FcR blocking. About 1×105 cells were placed into each of four tubes, and the antibodies below were added to the respective tubes and then mixed to produce isotype controls and samples for fluorescence correction. Each of the antibodies was added such that they were diluted 100 times.
Tube 1: FITC-labeled rat IgG2a, PE-labeled rat IgG2a, and biotinylated rat IgG2a
Tube 2: FITC-labeled anti-mouse CD45 monoclonal antibody, PE-labeled rat IgG2a, and biotinylated rat IgG2a
Tube 3: FITC-labeled rat IgG2a, PE-labeled anti-mouse CD45 monoclonal antibody, and biotinylated rat IgG2a
Tube 4: FITC-labeled anti-rat IgG2a, PE-labeled rat IgG2a, and biotinylated anti-mouse CD45 monoclonal antibody
Biotinylated anti-mouse PCLP1 monoclonal antibody, APC-labeled anti-mouse c-Kit monoclonal antibody, and FITC-labeled anti-mouse CD34 monoclonal antibody were added to the remaining cells such that they were each diluted 100 times, and were then mixed to prepare the sample. After adding the antibodies, the respective cells were left on ice for 30 minutes.
The isotype controls, fluorescence correction samples, and samples were each washed with ice-cooled 2% FBS/PBS. The isotype controls, fluorescence correction samples, and samples were each rediluted with streptavidin-APC diluted 50 times with 2% FBS/PBS, and then left in ice for 30 minutes. They were then washed with ice-cooled 2% FBS/PBS. Cells were diluted at 1×106 cells per 5 μL of 7-AAD, and then left at room temperature for five minutes. The cells were diluted in 2% FBS/PBS or diluted in PBS to a concentration of 2×106 to 5×106 cells/mL, and then transferred to a cell separator tube.
Using isotype controls and fluorescence correction samples, the sensitivity of each parameter of the cell separator was adjusted and fluorescence corrections were made. With reference to the fluorescence intensity of the isotype control, cell population in the sample that was CD34-positive and c-Kit-positive were gated, and gates were further developed within this gate according to PCLP1 expression intensity, with sorting gates set for the following three regions:
CD34-positive, c-Kit-positive, and PCLP1-positive;
CD34-positive, c-Kit-positive, and weakly PCLP1-positive; and
CD34-positive, cKit-positive, and PCLP1-negative.
The sorting gates were set such that when the CD34-positive and c-Kit-positive cell fraction was defined as 100%, the PCLP1-positive subfraction was approximately 16%, the weakly PCLP1-positive subfraction was approximately 13%, and the PCLP1-negative subfraction was approximately 71%. The samples were developed according to the fluorescence intensity of PCLP1 and cell size (FS peak). With reference to the fluorescence intensity of the isotype control, strongly PCLP1-positive, moderately PCLP1-positive, weakly PCLP1-positive, and PCLP1-negative regions were each gated.
The cells were fractionated into tubes containing 50 μg/mL gentamicin/15% FBS/DMEM mixed with 10 μg/mL OSM, 1 μg/mL bFGF, and 100 μg/mL SCF. The fractionated cells were reanalyzed to confirm that they were purely fractioned according to the gates that were set. The number of obtained cells was counted using a hemocytometer.
On a 10-cm dish or 6-well plate, OP9 stromal cells were cultured in 50 μg/mL gentamicin/15% FBS/DMEM until 70% to 90% confluent. Immediately before plating the sorted cells onto this dish or plate, the medium was replaced with that containing cytokines (10 μg/mL OSM, 1 μg/mL bFGF, and 100 μg/mL SCF). The following cells obtained by sorting were plated:
CD34-positive, c-Kit-positive, and PCLP1-positive fraction (2600 cells/well);
CD34-positive, c-Kit-positive, and weakly PCLP1-positive fraction (2600 cells/well);
CD34-positive, cKit-positive, and PCLP1-negative fraction (2600 cells/well); and
Strongly PCLP1-positive cells (2000 cells/well).
The cells were cultured in a CO2 incubator at 37° C. under 5% CO2 partial pressure conditions. Blood cell production in each of the fractions was observed under a microscope for several weeks, starting from the day after plating.
The expression of PCLP1 in the spleen could be categorized into four groups according to expression intensity:
Strongly positive (PCLP1++; approximately 1%);
Moderately positive (PCLP1+; approximately 30%);
Weakly positive (PCLP1 low; approximately 18%); and
Negative (PCLP1-; approximately 51%).
This pattern of PCLP1 expression was similar to the pattern of PCLP1 expression in the E14.5 fetal liver. On the other hand, the fraction of CD34-positive, c-Kit-positive cells was detected to be a distinct group constituting 5% or so of cells, and PCLP1 expression in this fraction was mostly negative; however, a slight distribution was observed in the weakly positive or positive regions.
Observation of OP9 co-cultures of each fraction under a phase-contrast microscope showed that the CD34-positive, c-Kit-positive, and PCLP1-negative fraction actively produced blood cell-like cells for the first two to three weeks of culture. Blood cell growth in the weakly PCLP1-positive fraction was somewhat weaker than for the strongly positive fraction, and the growth was even weaker in the PCLP1-positive fraction (
In the strongly PCLP1-positive fraction, formation of many endothelial cell-like colonies, which were morphologically similar to the endothelial cell-like colonies formed from an OP9 co-culture of strongly PCLP1-positive fetal liver cells, were observed on Day 10 of culture (
The results showed that strongly PCLP1-positive cells also exist in the spleen, although in low frequency, and that endothelial-like colonies are produced, which are morphologically similar to those produced by OP9 co-culture of strongly PCLP1-positive cells of the fetal liver. Further, as for fetal liver, blood cell growth for PCLP1-positive cells was activated later than for PCLP1-negative cells.
These results showed that during the developmental process of a fetus, as the site of hematopoiesis shifts from the AGM region, where adult-type hematopoiesis begins, to the liver and spleen, a strongly PCLP1-positive cell fraction and a moderately PCLP1-positive cell fraction are consistently present in each of the hematopoietic organs, and throughout the transition, the strongly PCLP1-positive cell fraction consistently includes a high frequency of endothelial precursor cells, and the moderately positive cells include hematopoietic stem cell-like juvenile cells that continuously produce blood cells for a long time.
PBS
70% Ethanol
50 μg/mL gentamicin/15% FBS/DMEM (GIBCO BRL)
ACK Buffer: produced by sterilizing the following stock buffers and then mixing them at an A' to B ratio of 9:1.
FcR blocker (Pharmingen)
Biotinylated anti-mouse PCLP1 monoclonal antibody (MBL)
Streptavidin magnet beads (Miltenyi Biotec)
SCF
bFGF
mOSM
OP9
Apparatuses
Dissection table, scissors, tweezers, Kimwipes, Falcon tubes, 1-mL syringes (Terumo), 18 G injection needles (Terumo), cell strainer (Falcon)
Auto MACS (Miltenyi Biotec)
CO2 incubator (SANYO)
Fifty C57BL/6j mice aged three months or more were anesthetized and then subjected to cervical dislocation. The mice were laid on their backs on a dissection table and sprayed thoroughly with 70% ethanol. Using a pair of scissors, an incision was made in the skin of the leg, and excessive fat and muscle was cut out. The joint was dislocated by holding the base of the leg with a pair of scissors, and the femur was extirpated and rubbed thoroughly using Kimwipes to remove unnecessary flesh. Both ends of the femur were cut using a pair of scissors, and a needle was attached to a syringe to draw in a suitable amount of medium. Using a pair of tweezers, the femur was held above a 50-mL Falcon tube containing medium. The tip of the needle was placed into the bone, and one push of the piston was used to push out the femur bone marrow.
The tube in which the bone marrow was collected was centrifuged at 1200 rpm for five minutes and the supernatant was discarded. 20 mL of ACK buffer was added and mixed by pipetting, and the mixture was left on ice for ten minutes. An equivalent amount of medium was added and mixed by pipetting. This was transferred to a 50-mL Falcon tube set with a cell strainer to remove excess tissues and unwanted particles, and centrifuged at 1200 rpm for five minutes. The supernatant was discarded, medium was added and mixed by pipetting, and this was centrifuged again at 1200 rpm for five minutes. The supernatant was discarded, 10.5 mL of medium was added, and the cells were suspended. The cell suspension was passed through a cell strainer. The number of cells was counted, and some were transferred to a separate tube and then stored. Ten μL of FcR blocker was added for every 1×107 cells/mL, and this was allowed to react on ice for 15 minutes. Anti-mouse PCLP1 antibody was added to a final concentration of 20 μg/mL, and was allowed to react on ice for 30 minutes. Medium was then added to fill the tube to 15 mL, and this was centrifuged at 1200 rpm for five minutes. The supernatant was discarded, medium was again added to fill the tube to 15 mL, and this was centrifuged at 1200 rpm for five minutes. The supernatant was discarded, and streptavidin-magnet beads were added at 4 beads/cell. This was left on ice for ten minutes, then medium was added. This was centrifuged at 1200 rpm for five minutes, the supernatant was discarded, and medium was added again. This was centrifuged at 1200 rpm for five minutes, the supernatant was discarded, the cells were suspended in PBS+5% FBS, and the cell suspension was passed through a cell strainer.
Cells were separated on AutoMACS by selecting the POSSELD2 program, and PCLP1-positive cells and PCLP1-negative cells were collected in separate Falcon tubes.
OP9 cells were seeded at 1×104 cells per well on a 6-well plate, and cultured overnight at 37° C. Cytokines (10 ng/mL of OSM, 100 ng/mL of SCF, and 1 ng/mL of bFGF) were added to the medium. PCLP1-positive cells, PCLP1-negative cells, and unseparated cells were each plated at 1×104 cells per well, and then cultured at 37° C.
One hundred femurs were extirpated from fifty C57BL/6J mice, and bone marrow cells were separated. The number of obtained bone marrow cells was 1.1×109 whole bone marrow cells. When PCLP1-positive cells were separated from the obtained bone marrow using AutoMACS, 2.6×106 PCLP1-positive cells were obtained. Whole bone marrow cells, PCLP1-positive cells, and PCLP1-negative cells were each co-cultured with OP9 stromal cells, but on Day 8 of culture only those wells seeded with PCLP1-positive cells were confirmed to form cobble-stones of hematopoietic stem cell growth and have endothelial precursor cell-like colonies (
The above-mentioned results showed that endothelial precursor cells exist in the bone marrow of individuals, although in low frequency, and that a cell population that differentiates into endothelial precursor cells can be separated using anti-PCLP1 monoclonal antibodies.
The hematopoietic stem cells obtainable by the present invention are useful for treating various blood diseases. Specific examples include leukemia and immunodeficiency. In such diseases the hematopoietic system of a patient is reconstructed by autotransplantation or allotransplantation of the hematopoietic stem cells obtained by the present invention to the patient, enabling radical cure of the above-mentioned diseases. The present invention enables amplification of hematopoietic stem cells in vitro, and since introduction of genes is highly possible during this process, the present invention thus provides very useful methods for stem cell transplantation and gene therapy for blood diseases.
On the other hand, vascular endothelial precursor cells obtainable by the present invention are useful for treating vascular diseases. Specific examples include arteriosclerosis obliterans and myocardial infarction. Such diseases may be radically cured by regenerating new blood vessels in place of obstructed arteries, and by regenerating damaged vascular endothelial cells to reestablish sufficient blood flow. Such attempts have been made in the past using bone marrow cells, however, since bone marrow cells include only a small number of vascular endothelial precursor cells and also include cells that may differentiate into bone, muscle, adipocytes, and such, risks have been pointed out regarding methods for direct transplantation of bone marrow cells. Since the present invention comprises the steps of isolating vascular endothelial precursor cells, and amplifying them by culturing the cells in vitro, it may enable selective transplantation of vascular endothelial cells. Suppression of angiogenesis by the in vitro culture system of vascular endothelial precursor cells of the present invention may also be a method useful for developing anticancer agents which have the effect of protecting against the malignant transformation of cancers.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2003-406527 | Dec 2003 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP04/16470 | 10/29/2004 | WO | 00 | 8/24/2007 |
