EP0736169A1 - Method for obtaining a cell population enriched in stem cells and compositions derived therefrom - Google Patents
Method for obtaining a cell population enriched in stem cells and compositions derived therefromInfo
- Publication number
- EP0736169A1 EP0736169A1 EP94928610A EP94928610A EP0736169A1 EP 0736169 A1 EP0736169 A1 EP 0736169A1 EP 94928610 A EP94928610 A EP 94928610A EP 94928610 A EP94928610 A EP 94928610A EP 0736169 A1 EP0736169 A1 EP 0736169A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- cells
- stem cells
- cell population
- cell
- hematopoietic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5002—Partitioning blood components
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
- G01N33/56972—White blood cells
Definitions
- This invention is in the field of isolation of cell populations enriched in hematopoietic stem cells.
- Mammalian hematopoietic (blood) cells provide a diverse range of physiological activities.
- Blood cells are divided into lymphoid, myeloid and erythroid lineages.
- the lymphoid lineages comprising B cells and T cells, provide for the production of antibodies, regulation of the cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, and the like.
- the myeloid lineage which includes monocytes, granulocytes, and megakaryocytes as well as other cells, monitors for the presence of foreign bodies, provides protection against neoplastic cells, scavenges foreign materials, produces platelets, and the like.
- the erythroid lineage provides the red blood cells, which act as oxygen carriers.
- stem cells progenitor populations
- stem cells progenitor populations
- Stem cells are capable of self-regeneration and may become lineage committed progenitors which are dedicated to differentiation and expansion into a specific lineage.
- a highly purified population of stem cells is necessary for a variety of in vitro applications and in vivo indications.
- Stem cells find, use in a variety of clinical settings including, but not limited to, regenerating the hematopoietic system of a host deficient in stem cells; re-engrafting stem cells in a chemically and/or radiation stem cell depleted host; producing various lineages of hematopoietic cells; detecting and evaluating growth factors relevant to stem cell self- regeneration; developing hematopoietic cell lineages; and assaying for factors associated with hematopoietic development. Stem cells are also a target for gene therapy to endow blood cells with novel properties.
- Stem cells are important targets for gene therapy, where the genes inserted into the stem cells promote the health of the individual into whom the stem cells are transplanted.
- the ability to isolate stem cells may serve in the treatment of lymphomas and leukemias, as well as other neoplastic conditions.
- Stem cells constitute only a small percentage of the total number of hematopoietic cells. Hematopoietic cells are identifiable by the presence of a variety of cell surface protein antigen "markers.” Such markers may be either specific to a particular lineage (lineage specific) or progenitor cell or be present on more than one cell type. Currently, it is not known how many of the markers associated with differentiated cells are also present on stem cells. One marker which was previously indicated as present solely on stem cells, CD34, is also found on a significant number of lineage committed progenitors. In particular, 80-90% of the CD34 + population is marked by lineage specific and non-specific markers.
- Table 1 summarizes probable phenotypes of stem cells in fetal, adult, and mobilized peripheral blood.
- myelomonocytic stands for myelomonocytic associated markers
- NK stands for natural killer cells
- AMPB adult mobilized peripheral blood.
- the negative sign or, uppercase negative sign, (") means that the level of the specified marker is undetectable above Ig isotype controls by fluorescence activated cell sorter (FACS) analysis, and includes cells with very low expression of the specified marker.
- FACS fluorescence activated cell sorter
- NK and T cell markers B cell Myelomonocytic Other P-gp markers Activity
- Negative selection is a process which depletes specific cell types to yield a cell population more highly concentrated in the population to be purified. Negative selection is usually insufficient to yield a suitable purified population of cells and must be followed by positive selection. Positive selection directly isolates the cells to be purified on the basis of cell specific characteristics. Positive selection technigues include, but are not limited to, FACS.
- Further physical separation technigues include, but are not limited to, magnetically stabilized fluidized bed such as that described in United States patent application serial No. 08/130,094.
- Other types of separation technigues utilize eguilibriu buoyant density sedimentation to separate cell types and cellular debris on the basis of their respective buoyant densities.
- Such separation technigues include, but are not limited to, centrifugation in Ficoll or Percoll.
- FACS can also be used to separate cells on the basis of physical characteristics.
- the type of light scatter of particular cells can be used to identify these cells for separation purposes.
- Low forward light scatter indicates small elements such as platelets and red blood cells.
- Forward light scatter increases with increasing cell size: lymphocytes, stem cells, monocytes, and granulocytes in that order.
- Side scatter is an indication of internal structure.
- Granulocytes which have high forward scatter because of their large size, also have high side scatter due to intracellular granules.
- Red blood cells having little internal structure, have low side scatter as well as low forward scatter.
- Characteristics other than cell surface markers are also used to isolate stem cells. Decreased rhodamine 123 (rhol23) staining of hematopoietic cells is determined not by the initial dye accumulation but by an efflux process sensitive to P-glycoprotein (P-gp) inhibitors. Retention of several P-gp-transported fluorescent dyes, including rhol23, in human bone marrow cells was found to be inversely correlated with the expression of P-gp. Bone marrow cells expressing physical and antigenic characteristics of pluripotent stem cells showed high levels of P-gp expression and fluorescent dye efflux. Fractions of human bone marrow cells isolated on the basis of either increased rhol23 efflux or P-gp expression contained practically all the primitive progenitor cells of human bone marrow, including
- lysosomal membrane releasing degradative enzymes, which cause lysis of the cells.
- Monocytes lyse immediately, whereas granulocytes do not.
- Lysosomotropic agents such as L-phenylalanine methyl ester hydrochloride (PME) have been shown to lyse human monocytes in vitro and have been used to selectively decrease the proportion of monocytes in mixtures of leukocytes.
- PME has been reported to be a "fugitive" reagent, selectively lysing monocytes without destroying the cytolytic function of lymphokine-activated killer cells (LAK cells) or NK cells.
- LAK cells lymphokine-activated killer cells
- NK cells cytolytic function of lymphokine-activated killer cells
- Certain L-amino acid lower alkyl esters have been found to increase the biological activity of LAK cells. Leung (1989) Cancer Immunol. Immunother. 30:247- 253; Wersall et al. (1990) Acta Oncol. £:431-437; Foon et al. (1992) J. Immunother. 1J : 184-190 ; and U.S. Patent No. 4,849,329.
- Elutriation cannot be immediately used for negative selection of white blood cells from peripheral blood or bone marrow because of the high red blood cell content of these tissues.
- the standard practice is to dilute the sample and subject it to equilibrium density gradient centrifugation on a Ficoll-diatrizoate density gradient.
- the mononuclear cells defined as a cell population containing hematopoietic cells as well as other cells with a single nucleus, band at the interface of the density gradient medium and the sample.
- the red blood cells and the mature granulocytes having greater densities by virtue of hemoglobin iron and dense granules respectively, sediment to the bottom of the tube.
- the remaining cells can then be further separated by other methods including, but not limited to, FACS, elutriation or affinity chromatography.
- FACS Fluorescence-Activated Cell Sorting
- Flow sorting of cells is described by Lindmo et al. (1990) Flow Cytometry and Sorting, 2 ed. Wiley Liss, Inc.
- mouse stem cells have been obtained in at least highly concentrated, if not a purified form, where fewer than about 30 cells obtained from bone marrow were able to reconstitute all of the lineages of the hematopoietic system of a lethally irradiated mouse. Each assayed cell is multipotent for all blood lineages, while self-renewal is variable amongst these cells.
- stem cell purification systems provide such a system.
- the invention provides methods for obtaining a cell population enriched in hematopoietic stem cells utilizing physical separation, the addition of at least one lysosomotropic agent, at least one purification step to remove cell debris and residual red blood cells, and a purification step to sort specifically for cells expressing stem cell specific markers but not lineage specific markers.
- the invention further provides a cell population enriched in stem cells obtained by the methods described herein.
- the present invention provides a method for obtaining a cell population substantially enriched in stem cells.
- stem cells are those hematopoietic cells expressing stem cell specific markers but not lineage specific markers.
- the method results in a high yield of stem cells which are capable of differentiating into the various hematopoietic lineages associated with stem cells and thus are not merely progenitor cells.
- the method comprises the steps of obtaining a hematopoietic cell population substantially free of red blood cells, physically separating the cells under conditions effective to obtain a cell population enriched in monocytes and stem cells, treating this cell population with at least one lysosomotropic agent at a concentration and under conditions sufficient to deplete monocytes and granulocytes and to retain stem cells, removing cell debris, and sorting the processed cells so as to obtain those cells expressing stem cell specific markers but not lineage specific markers.
- Hematopoietic cells can be obtained from a variety of sources including, but not limited to, adult bone marrow, adult peripheral blood, adult mobilized peripheral blood fetal bone marrow, fetal liver, fetal peripheral blood, umbilical cord blood and cadaveric bone marrow. Methods of obtaining these cells are known in the art and need not be described in detail herein. Although the invention is described with reference to human hematopoietic cells, it is to be understood that the invention applies to the corresponding cells of other mammals. Preferably, the invention is used with human cells and for the treatment of humans. Preferably, adult mobilized peripheral blood is used.
- the hematopoietic cells should be substantially free of red blood cells.
- substantially free of red blood cells means that the hematocrit is less than 5%. More preferably, the hematocrit is less than 2% of the cells.
- a peripheral blood preparation may be collected using a blood cell processor such as the Fenwall CS-3000 Cobe Spectrum or Hae oneties USO. At the time the blood is obtained from these instruments, it may be substantially free of red blood cells and no further processing may be necessary prior to initiating the method described herein. Further processing may be done while the blood is being collected or after collection. In the case of bone marrow cells, a processing step is generally required to remove excess red blood cells. Any method known in the art is suitable for this purpose including, but not limited to, equilibrium buoyant density sedimentation. Suitable methods are described in the Examples. Preferably, the method performed on the Stericell as described in the Examples is used.
- the hematopoietic cell population substantially free of red blood cells is then subjected to physical separation.
- Any method known in the art that is not lethal to stem cells is suitable for use. Suitable methods include, but are not limited to, elutriation and magnetically stabilized fluidized bed. Preferably, elutriation is used. Methods of performing physical separation are known in the art and are not described in detail herein. Elutriation is described in the Examples.
- the physically separated cell population is then treated with a lysosomotropic agent.
- agents include, but are not limited to, L-amino acid lower alkyl esters and hydrogen chloride salts thereof.
- Lower alkyl means an alkyl group of 1-4 carbon atoms. Carbon atoms include, but are not limited to, methyl and ethyl groups.
- L-amino acid lower alkyl esters suitable for use in the present invention are those which cause lysis of monocytes but which do not substantially affect viability of stem cells.
- the reagents also cause lysis of granulocytes.
- the reagents are relatively stable such that they do not form toxic compounds when placed in solution.
- the reagent used is L-phenylalanine methyl ester (PME) .
- PME L-phenylalanine methyl ester
- L-leucyl leucine methyl ester LLME
- LLME forms toxic polymers when placed in solution at concentrations over 0.05 mM. These polymers are toxic to all cell types and thus lyse not only monocytes but stem cells as well.
- L-amino acid lower alkyl esters are described in the art as not being particularly toxic to monocytes.
- L-glutamic acid methyl ester as being effective in lysing monocytes. Given the discussion and examples herein and known cell identification methods, it is within the skill of one in the art to determine which L-amino acid lower alkyl esters are effective to deplete monocytes and granulocytes and retain stem cells.
- lysosomotropic agent it is preferable to incubate the cells with the lysosomotropic agent under conditions sufficient to substantially deplete any granulocytes that may be present. This is because the kinetics of cell death differ for granulocytes and monocytes. Monocytes lyse fairly rapidly and completely in the presence of lysosomotropic agents. Granulocytes, having fewer lysosomes, undergo a slower cell death in response to lysosomotropic agents. Granulocyte death involves the formation of vacuoles and change in cell shape preceding cell lysis.
- the incubation conditions effective to lyse monocytes and granulocytes must be determined empirically for each lysosomotropic agent and are within the skill of one in the art.
- the ideal reaction conditions have been found to be 5 mM PME final concentration, incubation at room temperature for forty minutes and then at 37°C for 30 minutes. These conditions are optimal, a variety of other conditions are satisfactory. For instance, incubations at 4°C overnight and at room temperature for 2 hours and then at 4°C overnight yield results comparable to the optimal conditions.
- a Percoll separation is performed essentially according to the manufacturer's instructions. In this instance, the cells form a pellet and the cellular debris floats on the surface of the Percoll solution. The supernatant can be aspirated to yield a cell pellet enriched in stem cells.
- a step may be included to deplete the cell population of substantially any remaining mature red blood cells. This step may be performed by equilibrium density gradient banding of mononuclear cells or by incubation of cells with ammonium chloride.
- a final, sorting step then follows to yield cells expressing stem cell specific markers but not lineage specific markers.
- Any method known in the art is suitable for use including, but not limited to, FACS.
- the cells are sorted for expression of CD34 and lack of lineage specific markers (CD34 + Lin") .
- Lineage specific markers include, but are not limited to, CDIO, 19, 20, SIg for B-cells; 16, 33 for myeloid; 15 for granulocytes, 14 for monocytes, 41 for megakaryocytes, 38 for lineage dedicated, CD3, 4, 7, 8, TCR for T-cells and glycophorin for erythroid.
- the cells also express the Thy-1 antigen (CD34 + Thy-1 + Lin”) .
- the cells Prior to FACS sorting, the cells are stained with fluorescent antibodies specific for the particular markers.
- the following procedure is used to isolate stem cells from hematopoietic cell populations derived from either peripheral blood or bone marrow.
- peripheral blood samples with a hematocrit of greater than 5% the cell samples are sequentially subjected to FACS, Ficoll purification, elutriation, PME lysis, ammonium chloride lysis, and FACS sorting.
- peripheral blood samples with a hematocrit of less than 5% the cell samples are sequentially subjected to FACS, elutriation, PME lysis, Ficoll flotation and FACS.
- the cell samples are sequentially subjected to FACS, SteriCellTM purification for banding of mononuclear cells by equilibrium density sedimentation, elutriation, PME lysis, and FACS sorting.
- FACS Fluorescence Activated Cell Sorting
- FACS analysis was performed according to standard methods known in the art using antibodies specific for CD34, Thy-1, and lineage specific markers (14 * , 15"). Briefly, the following procedure was used. For FACS staining and analysis, 300 ⁇ L of each cell sample is used. The remainder of the cell samples were reserved for the isolation of stem cells. 1) A 3-well stain in a microtiter plate is used for STAT analysis. Into each of 3 wells, add 100 ⁇ L of cells by pipette. Centrifuge plate at 350 x g for 3 minutes and aspirate off the supernatant.
- FITC fluorescein isothiocyanate
- PE phycoerythrin
- TR Texas Red
- Bone marrow was obtained by aspiration and the white blood cells were counted. 1 x 10 8 cells are dispensed into labeled tubes and diluted to 20 mL with BW medium (BioWhittaker Elutriation Media with 0.5% HSA, pH 7.2). The platelets, red blood cells and plasma were removed from the sample using the SteriCellTM Processor (DuPont) according to the manufacturer's instructions and the fraction between 1.040 and 1.079 g/mL was reserved for subsequent purification steps.
- BW medium BioWhittaker Elutriation Media with 0.5% HSA, pH 7.2
- the chamber, chamber gasket, and all components of the Quick-Release Assembly which come into contact with the sample are sonicated for two minutes in a 1% solution of Liquinox and rinsed exhaustively with deionized water. The black seal is checked for scratches and irregularities, and, if necessary, polished by hand.
- the Quick-Release Assembly is assembled according to the manufacturer's directions with new 0-rings and new bearings being used in the assembly.
- the chamber is assembled according to the manufacturer's directions with the chamber screws untightened.
- the Quick-Release Assembly and the chamber are autoclaved separately. Assembly of Elutriation System
- Obtain a cell count on the sample Dilute 20 ⁇ L sample with 180 ⁇ L of saline.
- the target range for number of cells to introduce into the large Sanderson chamber is about 5 x 10 9 - 1 x 10 10 .
- the cell number is total cells, white cells plus red cells.
- Pump Calibration Have ready a 50 mL graduated cylinder, a timer, stopwatch, and a calculator with a linear regression program.
- the pump should be calibrated while the rotor is at run speed. Turn the centrifuge on if you have not already done so and, if necessary, enter the correction speed and temperature. Set the time for 59 hours, 59 minutes. Check to see that the Quick Release Assembly is installed correctly and that there are no large air bubbles in the chamber. Set the pump speed to 1.00 using the 10-turn helipot on the front of the pump and turn the pump on. Make sure the pump is pumping in the correct direction, i.e., from the reservoir to the rotor. If it is not, switch pump directions using the toggle switch on the pump. To avoid blowing a fuse on the pump, go from one pump direction to the other with a slight pause in the middle.
- loading is at 15 ml/min at 2500 rpm and then the flow rates for the large chamber at 2000 rpm are: El,
- E2 includes E2 and E3 from small chamber, 53 mL/min; and E3, R/O.
- a 10 mL syringe is a good size, but any size which is appropriate for your sample volume is fine.
- Example 5 Resuspend the cell pellet obtained from centrifuging the PME lysis suspension in 25% Percoll in ammonium chloride buffer which has been kept at 4°C. Use 50 mL of lysis buffer for each 10 8 cells. Incubate the cells for 5 minutes on ice. Centrifuge at 600 x g for 5 minutes to pellet the cells. Aspirate off the supernatant.
- Example 6 The following procedure was used to stain the cells obtained in Example 6 for subsequent FACS.
- the antibodies used correspond to commercially available antibodies. Resuspend the cells in Staining Buffer (MEM, 1% HSA, pH 7.4) containing Gammimmune (1:50) at a cell concentration of 2 x 10 7 . Cells and reagents must be kept cold from this point on. Add PR3-Fab'2-SR (anti-CD34- sulforhodamine labeled) , PR4-FITC, and PR9-FITC (anti-
- Anti-CD34 at 2.5 ⁇ g/mL was added at 6.5 ⁇ L/mL of cell suspension.
- the cell suspension in all cases was 2 x 10 7 /mL.
- the lineage-specific antibodies were at 1 ⁇ g/mL each and were added at 2 ⁇ L/mL of cell suspension.
- the anti-Thy antibody was 5 ⁇ g/mL and used at 6.5 ⁇ L/mL of cell suspension.
- Add PR13-b (biotin labeled) to a final concentration of 6.5 ⁇ g/mL to sample. Mix. Remove 50 ⁇ L aliquot for Gate tube 3. Place on ice covered with foil.
- FACS was performed according to standard methods known in the art using antibodies specific for CD34, Thy-
- Table 2 summarizes the results obtained from a series of experiments performed as described in Examples 1-7.
- HN and BM stand for bone marrow
- PB stands for mobilized peripheral blood
- 34L is the percentage of CD34 + Lin " cells in the original sample
- Tot34L is the actual number of CD34 + Lin " cells in the sample
- rec34L is the percent recovery of the CD34 + Lin” cells
- My is the percent mature myeloid cells in the original sample as determined by high orthogonal light scatter
- TotMy is the actual number of these mature myeloid cells in the sample
- DepMy is the percent depletion in the sample after the indicated series of steps
- 34LT is the percentage of CD34 + Thy-1 + Lin " cells in the original sample
- Tot34LT is the total number of
- the original percentage of stem cells was very low. Generally, if there are less than 1% CD34 + cells in the initial sample, the estimation of yields are approximate and subject to a high degree of error. It is clear from the results obtained that the present method yields a high percentage of the stem cells; in fact, close to 100% of the stem cells in the original sample can be recovered.
- the CD15 bead depletion was performed by methods known in the art. Briefly, 15 mL of HBSS with 2% HSA was added to anti-CD-15 antibody coated magnetic beads (roughly four times as many beads as granulocytes in the bone marrow sample) and the tube was placed in the magnetic stand. After 2 to 3 minutes, the buffer was carefully removed without disturbing the beads. The tube was removed from the magnetic stand and this wash step was repeated three additional times. The beads were resuspended in 1 mL of HBSS with 2% HSA and the bone marrow cells were resuspended in the same buffer at a concentration of 2 X 10 7 cells/mL.
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Abstract
Description
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Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US238686 | 1981-02-27 | ||
US12297793A | 1993-09-17 | 1993-09-17 | |
US122977 | 1993-09-17 | ||
US23868694A | 1994-05-05 | 1994-05-05 | |
PCT/US1994/010501 WO1995008105A1 (en) | 1993-09-17 | 1994-09-16 | Method for obtaining a cell population enriched in stem cells and compositions derived therefrom |
Publications (2)
Publication Number | Publication Date |
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EP0736169A1 true EP0736169A1 (en) | 1996-10-09 |
EP0736169A4 EP0736169A4 (en) | 1998-06-10 |
Family
ID=26821077
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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EP94928610A Withdrawn EP0736169A4 (en) | 1993-09-17 | 1994-09-16 | Method for obtaining a cell population enriched in stem cells and compositions derived therefrom |
Country Status (4)
Country | Link |
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EP (1) | EP0736169A4 (en) |
AU (1) | AU7798294A (en) |
CA (1) | CA2171951A1 (en) |
WO (1) | WO1995008105A1 (en) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5728581A (en) * | 1995-06-07 | 1998-03-17 | Systemix, Inc. | Method of expanding hematopoietic stem cells, reagents and bioreactors for use therein |
AU2003295444A1 (en) | 2002-11-15 | 2004-06-15 | The Board Of Trustees Of The University Of Illinois | Methods for in vitro expansion of hematopoietic stem cells |
JP2010525836A (en) | 2007-05-04 | 2010-07-29 | ホワイトヘッド・インスティテュート・フォー・バイオメディカル・リサーチ | Ex vivo expansion of human hematopoietic stem cells |
EP3382008A1 (en) | 2010-06-15 | 2018-10-03 | FUJIFILM Cellular Dynamics, Inc. | Generation of induced pluripotent stem cells from small volumes of peripheral blood |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
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US5061620A (en) * | 1990-03-30 | 1991-10-29 | Systemix, Inc. | Human hematopoietic stem cell |
-
1994
- 1994-09-16 CA CA 2171951 patent/CA2171951A1/en not_active Abandoned
- 1994-09-16 WO PCT/US1994/010501 patent/WO1995008105A1/en not_active Application Discontinuation
- 1994-09-16 AU AU77982/94A patent/AU7798294A/en not_active Abandoned
- 1994-09-16 EP EP94928610A patent/EP0736169A4/en not_active Withdrawn
Non-Patent Citations (2)
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No further relevant documents disclosed * |
See also references of WO9508105A1 * |
Also Published As
Publication number | Publication date |
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WO1995008105A1 (en) | 1995-03-23 |
CA2171951A1 (en) | 1995-03-23 |
EP0736169A4 (en) | 1998-06-10 |
AU7798294A (en) | 1995-04-03 |
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18D | Application deemed to be withdrawn |
Effective date: 19970930 |