JP2002523085A - Stem cells carrying FGF receptor on cell surface - Google Patents

Abstract

(57) Abstract: composition of substantially pure purified pluripotent stem cells, fibroblast growth factor (FGFR) ^{and, CD34 +, CD34 - lin -} , Thy-1 +, AC133 + or c Positive for both primitive phenotypes such as -kit ⁺ . A state that is an embryonic stem cell is also a phenotype that displays a primitive state. This population may further include endothelial cells such as TIE-1 ⁺ , TEK ⁺ , CD31 ⁺ , VE-Cadherin ⁺ or VEGFR ⁺ , or a sub-population thereof having another marker that displays stromal cells such as STRO-1 ^+. Can be defined by

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION The present invention relates to a novel phenotype of stem cells containing fibroblast growth factor (FGFR) on the cell surface and having a phenotype indicative of a primitive state. The invention further provides
It relates to a subpopulation having a phenotype displaying endothelial or stromal cells.

BACKGROUND development of the invention, remodeling, capacity of tissues and organs for the regeneration and repair is dependent on the presence of stem cells that form progeny differentiated by time division (also referred to as progenitor cells).
Stem cells are present in the epidermis, intestinal epithelium and hematopoietic system, and there is often indirect evidence of stem cells in mesenchymal tissue. In vitro and in vivo studies have shown evidence of osteoblastic precursor cells in bone marrow and other stromal cell preparations. However, the identification of cells in these tissues and their relationship to cells with typical stem cell characteristics has not yet been established.

[0003] Endothelial cells are part of normal bone marrow. Long-term cultures of human bone marrow cells comprise a complex mixture of stromal cells containing adipocytes, fibroblasts, endothelial cells, macrophages and smooth muscle cells. Endothelial cells and hematopoietic cells are believed to be derived from normal progenitor cells, hemangioblasts.

[0004] Cell surface molecules on a variety of cells and, in particular, hematopoietic cells, describe surface molecules (markers) that can be identified by a population of monoclonal antibodies, each differentiated population (CD) displaying the same cell reactivity. Have been given a CD display. CD labeling is specified at the regularly held international research conference on human leukocyte differentiation antigens. For example, the CD19 marker is specific for B cells and the CD33 marker is specific for bone marrow cells. At present, it is unclear how many markers related to differentiated cells are present on stem cells. One of the markers shown to be present on stem cells is CD34. However, this marker has also been found on progenitor cells directed to a significant number of cell lines. Another marker known and believed to be naturally present on stem cells, the "primitive" marker, is AC13.
3 (Yin et al, 1997; Buhring et al, 1999), Thy-1 (Murray et al, 1995) and c-kit (D'Arena et al, 1998).

[0005] A small number of circulating CD34 ⁺ hematopoietic stem cells are known to be present in peripheral blood. The major source of adult CD34 ⁺ hematopoietic stem cells is bone marrow, and the purpose of this small circulating CD34 ⁺ cell population is unknown. One explanation is that bone marrow cells are "leaky" and stem cells leak out, circulate, and return to the bone marrow. A second possibility is that the function of these circulating stem cells is to seed at sites such as the liver and spleen, which can serve as auxiliary sites for hematopoiesis in crisis.

[0006] Human CD34 ⁺ hematopoietic cells isolated from bone marrow, cord blood, and peripheral blood are heterogeneous populations containing hematopoietic stem cells. Recent evidence indicates that circulating CD34 ⁺ cells also contain endothelial stem cells, which can also circulate (Asahara et al.
, 1997; Nieda et al, 1997; Shi et al, 1998; Lin et al, 1998). Have shown that CD34 ⁺ cells isolated from peripheral blood can be incorporated into the endothelium of ischemic blood vessels in test animals. Purified cord blood CD34 ⁺ cells also give rise to pseudohemophilic factor-expressing endothelial cells in vitro, which provides further evidence of circulating progenitor endothelial cells (Nieda et al, 1997). In addition, bone marrow cell-derived CD34 ⁺ cells also contain transplantable stromal cells (Prockop, 1997; Pereira et al, 1998).

Recently, compelling evidence has been shown that human CD34 ⁺ progenitor cells from peripheral or umbilical cord blood transplanted into NOD / SCID immunodeficient mice gave rise to human stromal cells (Goan et al. , 1997). Human stromal cells express endothelial cell-specific vascular endothelial growth factor (VEGF) receptor-2 (KDR) and pseudohemophilic factors, indicating that they are endothelial-derived. Also, recent evidence indicates that injection of whole bone marrow cells into test mice results in donor-derived fibroblasts in a large number of non-hematopoietic tissues (Prockop, 1997; Pereira, 1998); Is present in the bone marrow. Since CD34 ⁺ has been shown to be expressed by bone marrow stromal progenitor cells (Simmons et al, 1991), these stromal progenitors are within the CD34 ⁺ progenitor population and may be present in the bone marrow .

[0008] Thus, the CD34 ⁺ progenitor cell population is a heterogeneous fraction containing hematopoietic, endothelial and stromal / fibroblast lineage progenitor cells. Furthermore, pluripotent mesenchymal stem cells have been shown to be present in the bone marrow microenvironment that can differentiate into cells of hematopoiesis, chondrogenesis, tendonogenesis, adipogenesis and myogenic cell lines (Majumdar et al, 1998). . There is circulating endothelial progenitor / stem cells and there is also recent literature indicating that stromal stem cells in the bone marrow serve as a source of continuous regeneration of cells in many non-hematopoietic tissues.
Common developmental precursors that give rise to both hematopoietic and endothelial cells have recently been identified (Suda et al, 1997; Choi et al, 1998; Caprioli et al, 1998).

[0009] Recent evidence also indicates that embryonic stem (ES) cells can give rise to endothelial cells (Hirashima et al, 1999).

[0010] Fibroblast growth factors (FGFs) can reinforce other factors that stimulate hematopoietic progenitor cell proliferation (Wilson et al, 1991; Quito et al, 1996; Allouche, 1995; Yuen et al)
, 1998, U.S. Patent No. 5,612,211 and U.S. Patent No. 5,817,773). It has also been shown that basic FGF (FGF-2) acts to antagonize cytokines that induce differentiation (Burger et al, 1994). In addition, a small amount of 10-100 pg
FGF-2 on the order of / ml induces a more primitive phenotype in human K562 leukemia cells.

It is very useful to be able to separate stem cells, which are precursor cells of endothelial and / or stromal cells. The more primitive the stem cells, the more useful in bone marrow transplants. In addition, endothelial stem cells and stromal stem cells, or stem cells that are precursors to both, find many uses in repairing damaged vasculature and in other conditions where endothelial and stromal cells need to be recruited. Will get.

[0012] SUMMARY OF THE INVENTION An object of the invention is to overcome the drawbacks of the prior art.

Another object of the present invention is to isolate a novel phenotype of stem cells.

[0014] It is a further object of the present invention to identify and isolate stem cells for use in stem cell transplantation.

[0015] Still a further object of the present invention is to identify and isolate endothelial and / or stromal stem cells.

A small population of cells having a “primitive phenotype” such as CD34 ⁺ or CD34 ⁻ lin ⁻ is isolated, which expresses a cell surface receptor for fibroblast growth factor (FGF). The group of cells carrying the FGF receptor (FGFR) is designated as FGFR ⁺ . The FGFR ⁺ primitive phenotype cell population has several unique properties:

(1) CD34 ⁺ FGFR ⁺ cells have low forward scatter (FSC) and low side scatter (S
SC) is mainly present in the region of the fluorescence-activated cell sorter profile. That is, the majority of cells in this population are very small and low in particle size. These small cells have a FS of the fluorescence-activated cell sorter profile that is not normally analyzed.
Although located in the C / SSC region, this region contains many dead and apoptotic cells. Interestingly, this region has recently been shown to be a site of the mesenchymal stem cell population (Zoher et al, 1997). CD34 ^- lin ^- FGFR ⁺ cells have FSC / SSC characteristics that are similar to CD34 ⁺ FGFR ⁺ cells. The CD34 ^- lin ^- FGFR ⁺ group also contains a significant number of higher FSC cells.

(2) CD34 ⁺ FGFR ⁺ cells are deeply dormant, a feature of the stem cell population. Does not grow in culture until 30-60 days after separation.

The FGFR ⁺ primitive phenotype population is a characteristic population of stem cells that are precursor cells for endothelium and / or hematopoiesis and / or stroma. The FGFR ⁺ primitive phenotype cell population can be obtained from either general circulation, bone marrow, cord blood or embryonic stem cells, and can form endothelial, blood and stromal cells depending on the needs at that time.

BRIEF DESCRIPTION OF THE DRAWINGS FIG . 1 summarizes the fourteen experiments of Example 1 in a bar graph and shows the percentage of CD34 ⁺ FGFR ⁺ cells expressing the indicated third antigen.

FIGS. 2A-2F are flow cytometry plots for Experiment 7 of Example 1. FIG.
FIG. 2A is enclosed to remove the area containing many debris and doublets.
FSC vs. SSC points for all events with plotted R1 region
Things. FIG. 2B shows the intensity of 7-aminoactinomycin D (7-AAD) staining.
It is a histogram showing a degree. Region R2 has been described to indicate live cells
. FIG. 2C plots the points of SSC versus CD34 gated on R1 AND R2.
It is a thing that I did. Region R3 is CD34⁺Described to indicate cells. Figure
2D plots FSC vs. SSC points gated on R1 AND R2 AND R3
Surviving CD34 in R1⁺Features of cell FSC / SSC
It shows the sign. FIG. 2E shows CD34 vs. FG gated on R1 AND R2.
It is a plot of FR points. Region R4 is CD34⁺FGFR⁺Show
It has been described for. FIG. 2F is gated on R1 AND R2 AND R4.
FIG. 14 plots the FSC vs. SSC points, which shows the viable CD34 in R1. ⁺ FGFR⁺It shows the characteristics of cells.

FIG. 3 is a bar graph showing the percentage of viable FGFR ⁺ cells co-expressing the specified antigen.

FIG. 4 is a bar graph showing the growth of various populations of cells in the presence or absence of FGF-2 or FGF-2 + VEGF.

[0024] DETAILED DESCRIPTION stem cells of the invention, when implanted, for example, hematopoiesis patients can no longer be produced in such as radiation therapy, it is possible to restore the production of endothelial and stem cells. FGFR ⁺ primitive phenotype cells from other cells in the body, preferably CD34 ⁺ FGFR ⁺ or CD3
By isolating 4 ^- lin ^- FGFR ⁺ , relatively pure stem cells can be obtained, preferably separated from mixed cells and other substances, so that the stem cells can be safely transplanted into a patient if necessary. I do.

[0025] The present invention unique separation cells, their CD34 ⁺ or CD34 ^- lin ^- by a retaining state and fibroblast growth factor receptor is separated from the other cells. Cells can be obtained from (Civin, U.S. Pat. No. 4,714,680, U.S. Pat. No. 4,965,204, U.S. Pat. No. 5,035,994 and U.S. Pat. No. 5,130,144, Tsukam
oto et al, US Pat. No. 5,750,397 and Loken et al, US Pat.
No. 137,809, all of which are incorporated herein by reference) can be separated by conventional techniques for cell separation. That is, for example, a CD34-specific monoclonal antibody or an FGFR-specific antibody can be immobilized on a column, magnetic beads, or the like. All cell populations are then passed through a column or added to magnetic beads. Those that remain bound to the column or can be separated magnetically later, but which are magnetically bound, are cells that contain a marker that is recognized by the antibody used. That is, if an anti-CD34 antibody is used, the resulting cell population is very rich in CD34 ⁺ cells. If FGFR antibodies are used, the resulting cell population is very rich in FGFR ⁺ cells. The cell population is then enriched for other markers by repeating the process using a solid phase to which antibodies against the other markers are bound.

Another method for sorting CD34 ⁺ FGFR ⁺ cells is by flow cytometry, most preferably a fluorescence-activated cell sorter (FACS) from Becton-Dickinson under the name FACScan or FACScalibur. . By this technique, cells having a CD34 marker on the cells are labeled with a specific fluorescent dye by an anti-CD34 antibody conjugated to the specific fluorescent dye or the like. Similarly, the FGFR marker of the cell is an anti-FGFR conjugated to another dye.
Each antibody is labeled with a different fluorescent dye. The stained cells are applied to an analyzer, and the flowing cells are excited with a fluorescent dye and irradiated with an argon laser beam that emits light. This emission is detected by a set of optical filters by a photomultiplier tube (PMT) of a fluorescent dye specific to the emission wavelength. The signal detected by the PMT is amplified by its own channel and is represented by the computer in a variety of different forms, such as a histogram, a stippling display or a contour display.
That is, fluorescent cells that emit at one wavelength respond to a specific fluorescent dye-labeling reagent, whereas non-fluorescent cells or fluorescent cells that emit at a different wavelength do not express this molecule and emit at other wavelengths. A molecule that responds to a fluorescent dye-labeling reagent that emits
This flow cytometry is also semi-quantitative in that it shows the amount of fluorescence displayed by the cells (fluorescence intensity). This correlates, in a relative sense, to the number of molecules expressed by the cell.

[0027] The flow cytometer also has the function of measuring non-fluorescent parameters such as the size of molecules and light scattered by cells upon irradiation with a laser beam. Cell size is usually a direct measurement. Light scatter PMTs detect light scatter by cells either forward (forward scatter; FSC) or right angle (side scatter; SSC). Both parameters are affected by other factors, FSC is a measure of size, while SSC is a measure of cellular complexity.

Preferably, the flow cytometer comprises one or more PMT emission detectors. Additional PMTs can detect other emission wavelengths and simultaneously detect one or more fluorescent dyes each on a separate channel. The computer allows analysis of each channel or correlation of each parameter with another parameter. Fluorescein isocyanate (FITC), which has an emission peak at 525 nm (green), and 575 nm (orange red)
R-phycoerythrin (PE) having an emission peak at 620 nm (red), propidium iodide (PI) having an emission peak at 620 nm (red), and 7-aminoactiomycin D (7-AAD) having an emission peak at 660 nm (green). ), R- having an emission peak at 670 nm (red).
Phycoerythrin Cy5 (PE-Cy5), which contains allophycocyanin (APC) having an emission peak at 655-750 nm (dark red).

[0029] These and other types of FACS analyzers may have the additional ability to physically separate different fractions by deflecting cells of different nature into separate containers.

[0030] Any other method of separating the starting population of CD34 ⁺ FGFR ⁺ cells, such as bone marrow, peripheral blood or cord blood, can also be used according to the present invention. The various subpopulations of the present invention can be separated in a similar manner.

The isolated cell group of the present invention can be used for treatment methods such as stem cell transplantation and the other treatment methods described below and other treatment methods obvious to those skilled in the art. For example, the isolated population of cells can be administered directly into a vein of a mammalian patient in need of a bone marrow transplant in an amount sufficient to rebuild the patient's hematopoiesis and immune system. The exact effective amount can be readily determined by one skilled in the art, and will, of course, vary with the particular condition being treated by the therapy. However, for many applications, an amount containing approximately the same number of stem cells as present in one-half to one liter of aspirated bone marrow is sufficient.

That is, from bone marrow or blood containing cells that are positive for both CD34 and fibroblast growth factor receptor, and preferably do not contain cells that are substantially not positive for both CD34 and fibroblast growth factor receptor. This human cell suspension can restore hematopoietic cell production to those who are deficient in hematopoietic cell production. The suspension of these separated cells is administered to a patient in need thereof in an amount sufficient to restore hematopoietic / endothelial / stromal cell production.

[0033] Patients in need of such production are those with a particular need for hematopoietic, endothelial or stromal cells. For example, a patient with vascular injury, a person with a genetic defect in hematopoiesis, stromal or endothelial cells such as collagen deficiency, adenosine deaminase deficiency or clotting factor deficiency. Circulating stem cells appear to selectively populate hematopoietic, endothelial or stromal cell damage / defect sites.

CD34⁺Other "primitive" phenotypic indices are also known. These include AC133 ⁺ (Buhring et al, 1994; Yin et al, 1997), Thy-1 (Murray et al, 1995) and
And c-kit (Buhring et al, 1994; D'Arena et al, 1998). Primitive
Another group of cells of the vesicle is CD34⁻lin⁻It is. In the present invention, preferred tools of the present invention
CD34 which is an example⁺FGFR⁺Cells have significant numbers of AC133 markers (approximately
64%), contains the Thy-1 marker (about 52%) or the c-kit marker (about 75%)
I got the knowledge. Many of these cells carry one or more primitive markers.

[0035] In addition, certain markers are known to be endothelial markers. these
Include VE-Cadherin (also referred to as CD144) (Vittet et al, 1996), TIE-
1 (also called TIE) (Suda et al, 1997), TEK (also called TIE-2) (Suda et al.
 al, 1997; Hamaguchi et al, 1999) and CD31 (also called PECAM) (Watt
 et al, 1993). A significant amount of each of these markers is CD34⁺FG
FR⁺It is also present in cells. Approximately 86% of this cell group co-administers VE-Cadherin-
About 70% co-express CD31; about 47% co-express TEK
About 33% co-express TIE-1. This data is CD34⁺FGFR ⁺ This shows that the cell group is a primitive cell group that is a precursor of endothelial cells. these
Subpopulations with different endothelial markers can be isolated and are part of the present invention.
You. Stromal cell markers such as SYRO-1 are also known as certain additional markers
(Gronthos et al, 1994). SYRO-1⁺CD34 of the present invention⁺F
GFR⁺A subpopulation of cells is a group of primitive cells that are precursors of stromal cells. these
This subpopulation with stromal cell markers is also part of the present invention.

The results for co-expression of additional markers were obtained from a fluorescence-activated cell sorter (FACS) analysis using antibodies specific for cell surface antigens. Antibodies are 3
Labeled with one or four different fluorescent dyes, indicating that 100% of the viable FGFR ⁺ Thy-1 ⁺ cells co-express VE-Cadherin and 97%
EFR ⁺ AC133 ⁺ cells co-express VE-Cadherin and have 91% viable F
GFR ⁺ AC133 ⁺ cells co-expressed Thy-1 and 67% of the viable FGFR ⁺ TEK ⁺ cells showed co-expressing Thy-1. Cell sorter methods using additional fluorescent dyes may allow to separate these cells into CD34 ⁺ FGFR ⁺ cell populations that also display two or more of the various other primitive or endothelial markers discussed above. . Similarly, other markers, such as vascular endothelial growth factor-receptor (VEGF-R) (also referred to as KDR), which are markers for endothelial cells, can be included in such assays. The existence of a subpopulation of CD34 ⁺ FGFR ⁺ Thy-1 ⁺ VEGF-R ⁺ cells can be predicted, and this method allows one of ordinary skill in the art to identify and isolate this subpopulation without undue experimentation. This subpopulation represents a group of progenitor cells that can develop into either or both hematopoietic and endothelial cell lines.

The isolated CD34 ⁺ FGFR ⁺ cells grow significantly slower in culture with a long time lag of 4-6 weeks. As shown in Example 3 and Table 5, the cells contained FGF-
Grow dependently. The prolonged period of dormancy is associated with the stem cell phenotype, indicating that these cells have stem cell proliferation characteristics.

[0038] For example, CD34 can be obtained by the method described above using a FACS analyzer.⁺FGFR⁺Fine
Although the cells can be separated substantially purely, i.e., as a substantially uniform population of cells,
Not necessarily the CD34 of the present invention⁺FGFR⁺Stem cell populations need not be substantially pure
Absent. For example, if the cell population is CD34⁺And FGFR⁺Or different primitive phenotype
FGFR with display⁺90% or more of human stem cells characterized by
It is. Another aspect of the invention relates to FGFRs that are substantially uniform for other markers. ⁺ Includes a subpopulation of primitive phenotypic cell populations. This can be one or more
Endothelial markers, one or more additional primitive markers and / or
Or FG that is also positive for one or more of any of the stromal cell markers
FR⁺Contains a subpopulation of primitive phenotype human stem cells, and is this subpopulation substantially homogeneous?
Or at least 90% of the cells are FGFR⁺A primitive phenotype, one or more of which
A composition that is positive for any of the above additional primitive markers. Ie
This subpopulation may be a substantially homogeneous population of cells or more than 90% of the population.
The vesicle is CD34⁺FGFR⁺(Or CD34⁻lin⁻FGFR⁺) And TIE-
1⁺Is a composition. This subpopulation may also be a substantially homogeneous population of cells, or
More than 90% of the cells are CD34⁺FGFR⁺(Or CD34⁻lin⁻ FGFR⁺) And CD31⁺And / or TEK⁺And / or VEG
FR⁺And / or any of VE-Cadherin and / or endothelial markers
Is a composition that is positive for Similarly, this subpopulation contains substantially uniform cells.
Group or more than 90% of the cells are CD34⁺FGFR⁺(Or CD
34⁻lin⁻FGFR⁺) And one such as AC133, Thy-1 and c-kit.
A composition that is positive for one or more other primitive markers. this
A subpopulation may also be a substantially homogeneous population of cells or more than 90% of the cells.
Is CD34⁺FGFR⁺(Or CD34⁻lin⁻FGFR⁺) And one more
Or a composition that is positive for more other primitive markers, and
A composition that is positive for one or more endothelial markers. This subsidiary
The population may also be a substantially homogeneous population of cells, or 90% or more of the cells may be C cells.
D34⁺FGFR⁺(Or CD34⁻lin⁻FGFR⁺) And CD3
4⁺FGFR⁺(Or CD34⁻lin⁻FGFR⁺) And STRO-1
A composition that is positive for one or more stromal markers. like this
Compositions are also included in the present invention.

For some applications of the invention, eg, bone marrow transplantation, the stem cell population
It may be a small percentage of the total number of cells to be administered. The remaining cells replicate
Filler cells, which are impossible cells, may be used. Alternatively, the rest
May be any of the other types of cells from which the cells of the invention were originally isolated.
Good. That is, for example, the invention also relates to any of the phenotypes of the invention, namely
, CD34⁺FGFR⁺, CD34⁻lin⁻FGFR⁺, CD34⁺FGF
R⁺Thy-1⁺, CD34⁺FGFR⁺TIE-1⁺, CD34⁺FGFR ⁺ CD31⁺, CD34⁺FGFR⁺VEGF-R⁺, CD34⁺FGFR ⁺ Thy-1⁺VEGF-R⁺And at least 20%.
Sub-populations with low purity also have remarkable features compared to the prior art, and
Many of the recommended uses still have the advantages of the present invention. Phenotype of the present invention
30%, 40%, 50%, 60%, 70% or 80% or more of any of the cells
Are also considered to be part of the present invention.

Another method of defining the composition of cells of the invention is a suspension of human cells, including multifunctional stem cells or endothelial stem cells, substantially free of mature lymphoid myeloid cells. The cells are substantially all of the FGFR ⁺ primitive phenotype and are substantially free of mature lymphoid myeloid cells.

Thus, although the present invention has been described primarily with reference to preferred embodiments of CD34 ⁺ FGFR ⁺ cells, the present invention also includes FGFR ⁺ cells having other indications of the primitive phenotype according to the present invention. Must be understood. For example, CD34 ⁻ cells that are negative for cell line markers (lin ⁻ ) are even more primitive than CD34 ⁺ cells (Zanjani et al, 1999). That is, the CD34 ^- lin ^- phenotype must also be understood as a primitive indication according to the present invention. Furthermore, when embryonic stem cells are used as a source of cells from which the cells of the invention are separated, by definition all such cells have a primitive phenotype. That is, FGFR ⁺ cells isolated from embryonic stem cell resources are cells that naturally have a primitive phenotype. In addition, other primitive markers such as AC133, Thy-1 and c-kit can also be used as markers for primitiveness. That is, from the broadest aspect, the present invention relates to the phenotype is FGFR ^+, but are not limited ^{to, CD34 +, CD34 - lin -} , phenotype displaying a primitive stage cells containing Embryonic stem cells, AC133 ⁺ , Thy-1 ⁺ and c-
Regarding the phenotype that is kit. The invention further relates to the subpopulations mentioned above.

Multifunctional FGFR ⁺ primitive phenotype stem cells, or any other phenotype of the present invention, have considerable market uses and include one or more of the following: (1) For bone marrow transplantation To

(2) For Targeted Delivery of Anti-Tumor Agent The endothelial stem cells of the present invention are used for the purpose of delivering a blood pressure stabilizer and an anti-tumor agent to a rapidly growing vascular bed associated with a tumor. Endothelial cells are long-lived and stem cells can be used to deliver blood pressure stabilizing / anti-tumor agents to the rapidly expanding vascular bed associated with tumors without affecting the stable endothelium of established blood vessels. it can.

(3) For Coating Valves and Instruments Used in Surgery The endothelial stem cells of the present invention are used for coating valves and implantable devices and can eliminate many of the coagulation problems currently associated with these devices. . Endothelial stem cells can be cultured from a particular individual so that valves, implantable devices, etc. can be coated with autologous endothelial cells. A panel of HLA-compatible endothelial stem cells can also be produced for these purposes.

(4) As a Genetic Engineering Vector Genetically engineered stem cells implanted in endothelium, bone marrow, or connective tissue stroma are long-lived, adenosine deaminase or clotting factors and t-PA that promotes thrombolysis
And other proteins can be secreted. Wild-type genes can be incorporated into endothelial stem cells by either homologous or random recombination. Normal cells that are allogeneic endothelial stem cells and have no genetic defects can be used for therapy.
Another indication for gene therapy is the introduction of a drug resistance gene, such as a multidrug resistance gene, that allows normal stem cells to be advantaged and selectively forced. Diseases other than diseases associated with endothelial cells, diseases associated with the deficiency of specific secreted products such as hormones, enzymes, interferons, and factors can also be treated. By employing an appropriate regulatory initiation region, when the production of a defective protein is normal, production in a cell type different from the cell type producing the protein is performed in parallel with the original production, and induced production of the defective protein is performed. Ribozymes, antisense or other messages can be inserted to inhibit specific gene production or susceptibility to disease.

(5) For Repair of Vascular Damage Sites Genetically engineered endothelial stem cells that secrete factors such as tissue plasminogen activator that help prevent restenosis after balloon angioplasty can be injected. Asahara et al. (1997) have shown that endothelial stem cells selectively implant at sites of vascular injury.

(6) For the separation of novel molecules important for stem cell and tumor cell ecology

(7) For the generation of endothelium, stroma and blood cells required at that time

The stem cells of the present invention can be expanded by long-term in vitro culture with minimal differentiation until needed. However, stem cells can produce blood cells when treated with appropriate hematopoietic growth and differentiation factors, or can form a tubular network structure with endothelial cell characteristics when treated with appropriate agents, or When treated with an appropriate reagent, fibroblast-like stromal cells can be formed. In addition, the cultured stem cells of the present invention are functionally capable of transmitting an antigen-specific immune response in vitro, relocating leukocyte hematopoietic organs, and extending the survival of animals with a disrupted hematopoietic system. It can mature into transforming blood cells.

The phenotype of the particular stem cells isolated according to the present invention is novel, and one of ordinary skill in the art can readily convert the stem cells of the present invention within the ranges described above and other ranges without undue experimentation. Can be used. A specific explanation of such use is Wagner et al.
No. 5,744,347.

The following materials and methods are applicable to all of the following examples.

Materials and Methods Raw Materials for Human Cells Fresh and frozen cells from bone marrow, cytokine-fixed peripheral blood or cord blood were obtained from donors after informed consent.

Bone marrow or cord blood samples were RPMI1 containing 4 volumes of 10% fetal calf serum (FCS)
Diluted with 640 medium. Mononuclear cells were subjected to Histopak ™ -1077 density gradient (Sig
ma Diagnostics, St. Louis, MO) and wash twice with PBS-citrate
(PBS containing 13.6 mmol / L sodium citrate, 1 mmol / L adenosine and 2 mmol / L theophylline).

The leukocyte-depleted blood transfusion samples are usually not subjected to Histopak®-1077 density gradient centrifugation, but are washed twice with PBS-citrate before the filtration step.

The PBS-citrate washed cells (from all raw materials) were filtered on a 40 μm nylon cell strainer (Falcon 2340, Becton-Dickinson, New Jersey). 2 mL of cells
Resuspended in PBS-citrate and overlaid on 3 mL PBS-citrate / bovine serum albumin (BSA) cushion to remove platelets and centrifuged at 200 g for 10 minutes at room temperature. (Thoma et al, 1994). Repeat this step once or 2
Repeated once.

If necessary, DNase was used to separate DNA from necrotic cell debris.

The immunomagnetic separation samples were enriched for CD34 ⁺ cells by one of the following methods:

(A) Magnetic activated cell sorting (MACS) column, uniform supermagnetic polystyrene beads (
An anti-CD34 antibody coated on Miltenyl Biotec, Auburn, CA) is used. Separation was performed on a MiniMACS instrument as described by the manufacturer (Miltenyl Biotec, Auburn, CA).

(B) Magnetic separation using the Dinal CD34 progenitor cell sorting method (Dynal AS, Oslo, Norway), also using an anti-CD34 antibody immobilized on microbeads.

In two techniques, magnetically labeled cells are separated from other cells by a magnetic field.

Antibodies and reagents The following antibodies were used for immunofluorescence staining: CD34-FITC, CD34-PE,
CD34-RPE-CY5, c-kit-PE, CD38-FITC, mouse IgG
-FITC, mouse IgG-PE, mouse IgG-RPE, mouse IgG2a, streptavidin-PE, and goat-anti-mouse-RPE were Daco A / S Glos
trup, Denmark). Thy-1-FITC and Thy-1-PE were obtained from Immunotech, France. CD31-FITC, mouse IgG
Biotin, CD34-APC and mouse IgG-APC are available from Caltag Lab.
oratories, Burlingame, CA). HLA-DR-FITC, mouse I
gG and goat IgG were purchased from Sigma (Sigma ™, St. Louis, MO) and CD3
4-APC, CD31-PE, mouse IgG-APC were obtained from Becton-Dickinson (
Becton-Dickinson, San Jose, CA). AC133-PE was obtained from Miltenyi Biotec, Auburn, CA. VE-Cadherin FITC is W.
Courtesy of Dr. A. Mueller, Cornell University, New York. TIE 1-FITC, TIE 1-PE, biotinylated TIE 1
And biotinylated TEK were kindly provided by Dr. T. Suda, Kumamoto University, Kumamoto, Japan. FGF-R1 antibodies were obtained commercially from Dr. McClean WL, Texas A & M University, Houston, Texas, or from QED Biosciences Inc. (San Diego, CA). Anti-FGF
-R1-FITC was purchased from QED or prepared in our laboratory. In this lab, anti-FGF-R1-APC was produced in this lab, purified, and conjugated to allophycocyanin (APC). The binding of the antibody to APC was determined by using Prozyme (Sazyme, Sa).
n Leandro, CA) using a Picolink ™ binding kit, PJ25C.

Cell Staining and Flow Cytometry MACS-Selected or Dinal-Selected CD34 ⁺ cells were transferred to PBS / 0.1% BS.
A / 0.01% NaN ₃ / aprotinin 20 μg / mL (PBS / BSA / NaN ₃ /
Aprotinin). Non-specific binding of immunoglobulin to the Fc receptor and cell surface was blocked with human IgG and, if appropriate, either mouse or goat IgG. Cells were incubated with the appropriate antibodies for 30 minutes on ice. After washing twice with PBS / BSA / NaN ₃ / aprotinin, the cells were washed with FITC,
FACS caribou flow cytometer (Becton-Dick) equipped with an argon laser to excite PE, RPE-CY5 fluorescent dye and helium-neon iodide
inson, San Jose, Calif.) with a time difference adjusted according to the manufacturer's instructions for the excitation of allophycocyanin (APC). Select from 30,000 to 150,000 CD34 ⁺ selected cells using Cellket software (Becton-Dickinson, San Jose, C.).
Analyzed using A). The dye, 7-aminoactinomycin (7-AAD) (Sigma,
St. Louis, MO) was used in some experiments to identify dead cells. this is,
This was performed to confirm that the CD34 ⁺ FGFR ⁺ cell group was a viable cell group.

[0063]Cell sorting Separation and CD34⁺FGFR⁺For studying the growth characteristics of cell populations, CD34 ⁺ Selected cells were obtained as described above. These cells were isolated from CD34 and FGF-R.
Incubate with antibody against 1 (above) and Coulter Epix Elite
(Beckman Coulter Inc., Fullerton, CA)⁺FGFR⁺And CD
34⁺FGFR⁻The cell group was sorted. The sorted cells were isolated from FGF-2 and VEG.
12.5% horse serum and 12.5% in the presence or absence of growth factors such as F
RPMI 1640, αMEM, DMEM and long-term culture medium with% FCS
(LTCM) (Myelocult from StemCell Technologies Inc., Vancouver, Canada
H5100). Count the numbers that exist at different times
Growth was assessed by determining

Example 1 CD34 ⁺ FGFR ⁺ co-expression of other antigens CD34 enriched cells were isolated using cytokine migrating peripheral blood (PB), bone marrow (BM), or umbilical cord using magnetic separation technology (Dynal or MiniMacs). Purified from blood (CD). Assays were performed on CD34 ⁺ FGFR ⁺ cells using fluorescently labeled antibodies and the percentage of CD34 ⁺ FGFR ⁺ was determined for each experiment. Table 2 shows the results of the 14 experiments performed. For 14 experiments, an average of 4.4% (± 2.3%) of CD34 ⁺ cells
GFR was expressed.

The presence of a third (third) antigen on CD34 ⁺ FGFR ⁺ cells was also confirmed using a fluorescent antibody. The average percentage of CD34 ⁺ FGFR ⁺ cells expressing a particular tertiary antigen is shown in Table 1 and in the graph of FIG. Error margins indicate standard deviation (SD), and numbers indicate the number of experiments in which a particular antigen was assayed.

The CD34 ⁺ or CD34 ⁻ lin ⁻ enriched cells were supplied to a Becton-Dickinson FACSCalibur device, and experiments were performed using CellQuest System software for three-color and four-color analysis. This technology is based on the Flow Cytometry Analysis Using the Becto
n-Dickinson FACScan ", Current Protocols in Immunology, units 5.4, 5.
It was similar to that described in 4.1-5.4, page 19 (Supplement 16, 1995).

[0067]

[Table 1]

It can be seen that in experiment 7, results were obtained for the third antigen from each panel of 9 different antigens. Table 2 summarizes the results of Experiment 7. In addition, flow cytometry plots for Experiment 7 are shown in FIGS. FIG. 2A plots forward scatter (ESC) versus side scatter (SSC) points for all events. The FSC gives an indication of cell size. That is, the smaller the FSC, the smaller the size. Granularity of cells by SSC
Alternatively, an index of complexity is provided. That is, the lower the SSC, the lower the particle size. Region R1 has been described to delineate low to high FSC and low to medium SSC cells. This eliminates the area containing most of the necrotic cell debris and doublets.

FIG. 2B is a histogram showing the intensity of staining with Dye 7-AAD (Philpott et al., 1996). 7-AAD is a macromolecule that is not easily taken up by cells with intact cell membranes, which strongly stain dead cells. This histogram is gated on R1, and region R2 is described as delineating live cells.

FIG. 2C plots the points of SSC versus CD34 gated on R1 AND R2. This gate is set using Boolean logic, according to which the convention used for R1 AND R2 is R1 * R2.
And in this example, this gate draws the viable cells into R1. Region R3 is described as delineating CD34 ⁺ cells in this plot. CD34 ⁺ cells were labeled using FITC.

FIG. 2D shows F gated to R 1 AND R 2 AND R 3 (R 1 * R 2 * R 3).
It is a plot of points of SC vs. SSC. That is, this plot uses "backgating" to show the FSC / SSC properties of live CD34 ⁺ cells in R1.

FIG. 2E is a plot of CD34 vs. FGFR points gated on R1 AND R2. That is, it is gated on living cells in R1. CD34 ⁺ F
Region R4 is described to delineate GFR ⁺ cells.

FIG. 2F shows the F gated to R 1 AND R 2 AND R 4 (R 1 * R 2 * R 4).
It is a plot of points of SC vs. SSC. That is, this plot shows the characteristics of live CD34 ⁺ FGFR ⁺ cells in R1 using “backgating”. It is noted that most viable CD34 ⁺ FGFR ⁺ cells have low FSC and low SSC, ie, they are small cells with low particle size. The location of these cells on the plot of scattered light points is somewhat unusual because they are represented in areas that are normally ignored.

[0074]

[Table 2]

Table 3 shows the results of some samples from Experiment 8. These samples do not contain CD34 staining so that other combinations of antigens can be assayed. FIG. 3 illustrates these results. All surviving FGFR ⁺ Thy-1 ⁺ cells had 97
It can be seen that VE-Cadherin is co-expressed as well as% viable FGFR ⁺ AC133 ⁺ cells. In experiment 8, 99% of CD34 ⁺ FGFR ⁺ cells had VE-C
Since it is found that Adherin ^+, from this ^experiment, CD34 + FGFR ⁺ that phenotype populations ^{CD34 + FGFR + VE-Cadherin +} Thy-1 + AC133 is ^+, and about two-thirds are TEK ⁺ You can see that.

[0076]

[Table 3]

Example 2: CD34 ^- lin ^- FGFR ⁺ co-expression of other antigens The following experiment was performed using the technique described in Example 1. Lineage depletion to obtain lin ^- cells was performed using Dynal beads and magnetic separation, as described in Bachia et al., 1997 and Bachia et al., 1998. The results obtained are summarized in Table 4. 77% of CD34 ^- lin ^- FGFR ⁺ cells have C
D31 coexpressed, 40% of CD34 ^- lin ^- FGFR ⁺ cells co-expressing c-kit, 47% of the CD34 ^- lin ^- FGFR ⁺ cells co-expressing VE-Cadherin, and 18% of CD34 ^- It can be seen that lin ⁻ FGFR ⁺ cells co-express TIE-1.

[0078]

[Table 4]

[0079]Example 3: Growth characteristics Cytokine-migrating peripheral blood cells were converted to CD34 using magnetic separation.⁺About the cells
And enriched. The cells were purified using fluorescently labeled antibodies against CD34 and FGFR.
Stain and use Fluorescent Cell Analyzer (FACS) for FGFR⁺Population (cell group) and FGFR ⁻ Classify into groups. Cells were either (a) free or (b) 10 ng / ml FGF-2, (c)
Long-term culture medium containing either 10 ng / ml FGF-2 + 10 ng / ml VGEF
1 well in collagen / gelatin coated well containing (LTCM)
And incubate at 37 ° C in a humidified incubator.
I did it. After 4 to 6 weeks, the number of cells per well was counted. The results are shown in Table 5.
Then, a graph is shown in FIG. FGFR⁺The population grows FGF-dependent and VE
It can be seen that the addition of GF and FGF-2 further increases cell proliferation.

[0080]

[Table 5]

All references (magazines or abstracts, published or unpublished,
(Including U.S. or foreign patent applications, U.S. or foreign issued patents) or other references, and are incorporated herein by reference, including data, tables, and figures in the cited references. And sentences. Further, the contents of all references cited in the references cited herein are also entirely described in the specification by reference.

Reference to a known method step, conventional method step, known method or conventional method refers to any aspect, description or embodiment of the invention that is disclosed, taught or suggested in the relevant art. I do not admit it.

The above description of particular embodiments fully illustrates the general nature of the present invention, so that anyone can apply their current knowledge without undue experimentation and Various embodiments can be readily modified and / or adapted without departing from the general concept, and therefore such adaptations and modifications may be made in accordance with the disclosed embodiment. It is to be understood that they are encompassed within the meaning and scope equivalent to and are intended to be so. It is to be understood that the terminology or terms used herein are descriptive and not limiting. The means, materials, and steps for performing the various disclosed functions may employ various alternatives without departing from the invention. Accordingly, it may be found in the above specification and / or the following claims after the functional description.
The terms "means for" and "means for ..." or the language of any method steps are not exactly equivalent to the embodiments disclosed in the above specification. Defines and encompasses any structural, physical, chemical or electrical element or structure, or any method step, present or future to perform the described function, whether or not Intended to be. That is, other methods or steps for performing the same function may be used, and such expressions are intended to be interpreted in the broadest sense.

[Brief description of the drawings]

FIG. 1 is a bar graph showing 14 experimental results of Example 1. Third (3)
(%) CD34 ⁺ FGFR ⁺ cells expressing antigen.

FIGS. 2A-2F are flow cytometry plots for Experiment 7 of Example 1. FIGS. FIG. 2A plots the R1 region and FSC vs. SSC points.

FIG. 2B is a histogram showing the intensity of 7-aminoactinomycin D (7-AAD) staining. Region R2 indicates a viable cell.

FIG. 2C is a plot of the gated SSC vs. CD34 points for R1 AND R2. Region R3 indicates CD34 ⁺ cells.

FIG. 2D is a plot of FSC versus SSC gated on R1 AND R2 AND R3. Figure 4 shows FSC / SSC characteristics of surviving CD34 ⁺ cells in R1.

FIG. 2E is a plot of CD34 vs. FGFR points gated on R1 AND R2. Region R4 represents CD34 ⁺ FGFR ⁺ .

FIG. 2F is a plot of FSC versus SSC gated on R1 AND R2 AND R4. Shows characteristics of live CD34 ⁺ FGFR ⁺ cells in R1.

FIG. 3 is a bar graph showing the percentage of viable FGFR ⁺ cells co-expressing a designated antigen.

FIG. 4 is a bar graph showing the growth of various cell populations in the presence or absence of FGF-2 or FGF-2 + VEGF.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 48/00 A61P 35/00 A61P 7/00 41/00 35/00 C12N 5/00 E 41/00 A61K 37/24 (72) Inventor Patricia E. Burger South Africa 7441 Cape Town, Blueburg Strand, Spanish Steps Ferguson Road 9th F-Term (Reference) 4B065 AA93X AC14 AC20 BA21 BA24 BB19 BB34 CA44 CA46 4C084 AA02 AA13 BA44 DB54 MA01 NA14 ZA512 ZB212 ZB262 4C087 AA01 AA02 BB34 BB44 BB59 BB65 CA04 MA01 NA14 ZA51 ZB21 ZB26

Claims (28)

[Claims] 1. A composition comprising a physiologically acceptable medium and human stem cells, wherein at least 20% of said cells are FGFR ⁺ and have another indication of their primitive state. A composition comprising human stem cells. 2. The method according to claim 1, wherein the indication of the primitive state is selected from the group consisting of CD34 ⁺ , CD34 ⁻ lin ⁻ , embryonic stem cells, Thy-1 ⁺ , AC133 ⁺ and c-kit ⁺ . Composition. 3. The composition of claim 2, wherein said indication of the primitive state is CD34 ⁺ . Wherein said display primitive state, CD34 ^- lin ^- is, claim 2
A composition as described. 5. The composition of claim 2, wherein said indication of a primitive state is a state of embryonic stem cells. 6. The composition according to claim 1, wherein said human stem cells are selected from the group consisting of endothelial stem cells, stromal stem cells and hematopoietic stem cells. 7. 20% or more of the cells are FGFR ⁺ , either CD34 ⁺ or CD34 ⁻ lin ⁻ , and the markers Thy-1 ⁺ , AC1
33 is a human stem cell characterized in that it has a ⁺ and c-kit ⁺ 1 one or more composition of claim 1. 8. The method according to claim 8, wherein at least 20% of the cells are FGFR ⁺ , either CD34 ⁺ or CD34 ⁻ lin ⁻ and have one or more markers that indicate endothelial cells. 2. A human stem cell, which is characterized in that:
A composition as described. 9. The marker for displaying endothelial cells is TIE-1⁺, TEK ⁺ , CD31⁺, VE-Cadherin⁺And VEGFR⁺Selected from the group consisting of
9. The composition of claim 8, wherein the composition is 10. The method according to claim 10, wherein at least 20% of the cells contain FGFR.⁺And CD34 ⁺ Or CD34⁻lin⁻And one that displays stromal cells
Or a human stem cell, characterized in that it has a marker or more.
The composition of claim 1. 11. The composition according to claim 10, wherein the marker indicating the stromal cells is STRO-1 ⁺ . 12. The method according to claim 12, wherein at least 20% of the cells are FGFR.⁺And CD34 ⁺ Or CD34⁻lin⁻One that further displays endothelial cells
Or a human stem cell, characterized in that it has a marker or more.
7. The composition according to 7. 13. The method according to claim 13, wherein at least 20% of the cells are FGFR.⁺And CD34 ⁺ Or CD34⁻lin⁻And one that displays stromal cells
Or a human stem cell characterized by having more markers
8. The composition of claim 7, wherein the composition is characterized. 14. The composition of claim 1, wherein said human stem cells are a subpopulation of peripheral blood cells, bone marrow cells or cord blood cells. 15. The human stem cell is a subpopulation of embryonic stem cells.
A composition as described. 16. The human stem cell according to claim 1, wherein 90% or more of the cells are FGFR ⁺ and have another phenotype that indicates a primitive state.
A composition as described. 17. The FGFR comprises at least 90% of the cells.⁺And CD34 ⁺ Or CD34⁻lin⁻And the marker Thy-1⁺, AC
133⁺And c-kit⁺A person having one or more of the following:
The composition according to claim 1, which is a stem cell. 18. The method according to claim 18, wherein 90% or more of the cells are FGFR.⁺And CD34 ⁺ Or CD34⁻lin⁻And a marker that displays endothelial cells.
Claims: 1. A human stem cell having one or more of the cells.
The composition of claim 1. 19. The marker for indicating the endothelial cell may be TIE-1 ⁺ , TE
19. The composition of claim 18, wherein the composition is selected from the group consisting of K ⁺ , CD31 ⁺ , VE-Cadherin ^+, and VEGFR ⁺ . 20. More than 90% of the cells are FGFR⁺And CD34 ⁺ Or CD34⁻lin⁻And one that displays stromal cells
Or a human stem cell, characterized in that it has a marker or more.
The composition of claim 1. 21. The composition according to claim 20, wherein the marker indicating the stromal cell is STRO-1 ⁺ . 22. More than 90% of the cells are FGFR⁺And CD34 ⁺ Or CD34⁻lin⁻One that further displays endothelial cells
Or a human stem cell, characterized in that it has a marker or more.
7. The composition according to 7. 23. 90% or more of the cells are FGFR⁺And CD34 ⁺ Or CD34⁻lin⁻And one that displays stromal cells
Or a human stem cell characterized by having more markers
8. The composition of claim 7, wherein the composition is characterized. 24. The composition of claim 16, wherein said human stem cells are a subpopulation of peripheral blood cells, bone marrow cells or cord blood cells. 25. The human stem cell is a subpopulation of embryonic stem cells.
6. The composition according to 6. 26. A substantially homogeneous population of cultured human stem cells exhibiting the FGFR ⁺ phenotype and another phenotype indicative of a primitive state, said cells comprising endothelial cells, stromal cells and hematopoiesis. A cell composition selected from the group consisting of lineage cells and capable of generating cells. 27. A human cell suspension comprising pluripotent stem cells substantially free of mature lymphocytes and bone marrow cells. 28. The suspension of claim 27, wherein said stem cells are selected from the group consisting of endothelial cells, stromal cells and hematopoietic cells.