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WO1996015259A2 - Methode de mesure de cellules souches a lignage multiple et son procede d'utilisation - Google Patents

Methode de mesure de cellules souches a lignage multiple et son procede d'utilisation Download PDF

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Publication number
WO1996015259A2
WO1996015259A2 PCT/EP1995/004480 EP9504480W WO9615259A2 WO 1996015259 A2 WO1996015259 A2 WO 1996015259A2 EP 9504480 W EP9504480 W EP 9504480W WO 9615259 A2 WO9615259 A2 WO 9615259A2
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Prior art keywords
cells
stem cells
cell
stem
interleukin
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PCT/EP1995/004480
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English (en)
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WO1996015259A3 (fr
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Amittha Wickrema
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Novartis Ag
Novartis Erfindungen Verwaltungsgesellschaft Mbh
Systemix, Inc.
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Priority to AU41167/96A priority Critical patent/AU4116796A/en
Publication of WO1996015259A2 publication Critical patent/WO1996015259A2/fr
Publication of WO1996015259A3 publication Critical patent/WO1996015259A3/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • This invention provides methods for determining the presence of hematopoietic stem cells in a population of hematopoietic cells.
  • Mammalian hematopoietic cells provide a diverse range of physiologic activities. These cells are divided into lymphoid, myeloid and erythroid lineages.
  • the lymphoid lineage comprising B cells and T cells, provides 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, 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 are capable of self-regeneration but may also divide into progenitor cells that are no longer pluripotent and capable of self-regeneration. These progenitor cells divide repeatedly to form more mature cells which eventually become terminally differentiated to form the various mature hematopoietic cells. Thus the large number of mature hematopoietic cells is derived from a small reservoir of stem cells by a process of proliferation and differentiation.
  • stem cells refers to hematopoietic stem cells and not stem cells of other cell types.
  • progenitor or progenitor cells indicates cell populations which are no longer stem cells but which have not yet become terminally differentiated.
  • lymphoid, myeloid or erythroid in conjunction with progenitor indicates the potential cell populations into which the progenitor is capable of maturing.
  • a highly purified or enriched population of stem cells is necessary for a variety of in vitro experiments and in vivo indications. For instance, a purified population of stem cells will allow for identification of growth factors associated with their self-regeneration. In addition, there may be as yet undiscovered growth factors associated with: (1) the early steps of dedication of the stem cell to a particular lineage; (2) the prevention of such dedication; and (3) the negative control of stem cell proliferation.
  • Stem cells find use in: (1) regenerating the hematopoietic system of a host deficient in any class of hematopoietic cells; (2) a host that is diseased and can be treated by removal of bone marrow, isolation of stem cells and treatment with drugs or irradiation prior to re- engraftment of stem cells; (3) producing various hematopoietic cells; (4) detecting and evaluating growth factors relevant to stem cell self-regeneration; and (5) the development of hematopoietic cell lineages and assaying for factors associated with hematopoietic development.
  • Stem cells are also important targets for gene therapy, where expression of the inserted genes promotes 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 where the stem cells are purified from tumor cells in the bone marrow or peripheral blood, and reinfused into a patient after myelosuppressive or myeloablative chemotherapy.
  • Stem cells and progenitor 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 or carbohydrate "markers.” Such markers may be either specific to a particular lineage or be present on more than one cell type. The markers also change with stages of differentiation. Currently, it is not known how many of the markers associated with differentiated cells are also present on stem and progenitor cells. One marker which was previously indicated as present solely on stem cells, CD34, raised against KGla cells, is also found on a significant number of lineage committed progenitors.
  • U.S. Patent No. 4,714,680 describes a composition comprising human CD34 * stem and progenitor cells.
  • the CD34 marker is found on numerous lineage committed hematopoietic cells. In particular, 80-90% of the CD34 * population is marked by other lineage specific and non ⁇ specific markers. Therefore, in view of the small proportion of the total number of cells in the bone marrow or peripheral blood which are stem cells, the uncertainty of the markers associated with the stem cell as distinct from more differentiated cells, and the general difficulty in assaying for human stem cells biologically, the identification and purification of stem cells has been elusive. Characterizations and isolation of human stem cells are reported in: Bau et al. (1992) Proc. Natl . Acad. Sci . USA 59:2804-2808; and Tsukamoto et al. U.S. Patent No. 5,061,620.
  • Hematopoietic cells can be characterized by the level of rhodamine 123 (rhol23) staining, which 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 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, i.e. are rhodamine low.
  • rhol23 rhodamine 123
  • the mouse stem cell has 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 hematopoietic lineages, while self- renewal is variable amongst these cells.
  • the SCID-hu bone model allows the detection of myeloid and B-lymphoid cell potential in a putative stem cell population.
  • the HLA of the population which is injected is mismatched with the HLA of the host bone cells.
  • Stem cells from human hematopoietic sources have been found to engraft and sustain B lymphopoiesis and myelopoiesis in the SCID-hu bone model.
  • the SCID-hu thymus model allows the detection of T-cell potential in a test population.
  • fetal thymus is isolated and cultured for from 4-7 days at about 25°C, so as to deplete substantially the lymphoid population.
  • the cells to be tested for T cell activity are then microinjected into the thymus tissue, where the HLA of the population which is injected is mismatched with the HLA of the thymus cells.
  • the thymus tissue may then be transplanted into a scid/scid mouse as described in U.S. Patent No. 5,147,784, particularly transplanting under the kidney capsule.
  • assays of the thymus fragments injected with the test cells can be performed and assessed for donor-derived T cells.
  • the long-term culture initiating cell (LTC-IC) limiting dilution assay requires an initial 5 week stromal cell coculture followed by 2 weeks of methyl cellulose culture to obtain colony frequencies. This lengthy time period is required because progenitor cells read out at earlier time points whereas the colonies counted at 7 weeks are believed to be derived from stem cells.
  • the second limiting dilution assay measures the frequency of cobblestone area forming cells (CAFC) on a layer of stromal cells after 5 weeks of culture.
  • CAFC cobblestone area forming cells
  • the present invention encompasses methods of detecting and quantitating hematopoietic stem cells in a population of hematopoietic cells.
  • a population of hematopoietic cells is first obtained and treated to enrich for stem cells.
  • Single cells in the enriched population are deposited onto a suitable growth substrate such as a well in a 96 well microtiter plate.
  • the single cell deposits are cultured under conditions suitable to maintain viability and induce differentiation.
  • the resultant populations are then assayed for the expression of lineage-specific messenger RNA transcripts (hereinafter "transcripts”) specific to at least two different lineages. The presence of transcripts specific to two different lineages is scored as positive for the initial presence of a stem cell.
  • transcription lineage-specific messenger RNA transcripts
  • Figure 1 is a plot depicting the capillary electrophoresis results of a reverse transcription coupled polymerase chain reaction sample for the CD19 transcripts.
  • the present invention relates to methods of detecting and quantitating stem cells in a population of hematopoietic cells enriched for stem cells.
  • the cells are deposited as single cells on suitable stromal cells and cultured under conditions suitable to induce multilineage differentiation of stem cells.
  • the resulting cells are analyzed for the presence of mRNA transcripts of proteins from at least two different lineages.
  • the assay is completed in 2-3 weeks and does not rely on subjective analysis of cobblestone area cell morphology to determine the stem cell activity. This analysis is extremely sensitive and has the capability for analysis of large numbers of samples in a relatively short time.
  • the frequency of stem cells in the sample is calculated from the number of wells positive for transcripts from at least two different lineages. Since only primitive stem cells have pluripotential capability, results of this assay reflect the stem cell content of the original sample.
  • enrichment for stem cells relies on positive selection for cells bearing surface markers present on stem cells and/or negative selection to remove cells bearing surface markers absent on stem cells.
  • Stem cells have been characterized by the following phenotypes.
  • fetal cells including, but not limited to: CD34 ⁇ CD34 h ⁇ CD3 ' , CD7 ' ; CD8 " , CD10 ⁇ CD14 “ , CD15” , CD19 “ , CD20 “ , and Thy-1 * .
  • adult cells including, but not limited to: CD34 * , CD34 hl , CD3 “ , CD7 “ , CD8, CD15 “ , CD19 “ , CD20 “ , and Thy-1 * or as represented in Table 1.
  • rhol23 can divide the cells into high and low subsets ("rho 10 " and "rho hi ").
  • the cells are rho 10 .
  • the stem cells are human but may derive from any suitable animal.
  • LIN " cells generally refer to cells which lack markers associated with T cells (such as CD2, 3, 4 and 8), B cells (such as CD10, 19 and 20), myeloid cells (such as CD14, 15, 16 and 33), natural killer (“NK”) cells (such as CD2, 16 and 56), RBC (such as glycophorin A), megakaryocytes, mast cells, eosinophils or basophils.
  • 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 FACS analysis, and includes cells with very low expression of the specified marker.
  • Sources of cells for subsequent purification include, but are not limited to, bone marrow, both adult and fetal, mobilized peripheral blood (MPB) , blood, umbilical cord blood, embryonic yolk sac, fetal liver, and spleen, both adult and fetal.
  • Bone marrow cells may be obtained from any known source, including but not limited to, ilium (e.g. from the hip bone via the iliac crest) , sternum, tibiae, femora, spine, or other bone cavities.
  • an appropriate solution may be used to flush the bone, including but not limited to, salt solution, conveniently supplemented with fetal calf serum (FCS) or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from about 5-25 mM.
  • Convenient buffers include, but are not limited to, Hepes, phosphate buffers and lactate buffers. Otherwise, bone marrow may be aspirated from the bone in accordance with conventional techniques.
  • Various treatments may be employed to separate the cells.
  • cells of dedicated lineage are removed initially.
  • Monoclonal antibodies are particularly useful for identifying markers associated with particular cell lineages and/or stages of differentiation.
  • the antibodies may be attached to a solid support to allow for crude separation.
  • the separation techniques employed should maximize the retention of viability of the fraction to be collected.
  • Various techniques of different efficacy may be employed to obtain "relatively crude” separations. Such separations are where up to 10%, usually not more than about 5%, preferably not more than about 1%, of the total cells present not having the marker may remain with the cell population to be retained.
  • the particular technique employed will depend upon efficiency of separation, associated cytotoxicity, ease and speed of performance, and necessity for sophisticated equipment and/or technical skill.
  • Procedures for separation may include but are not limited to, magnetic separation using antibody-coated magnetic beads; affinity chromatography,* cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, including but not limited to, complement and cytotoxins; and "panning" with antibody attached to a solid matrix, e.g., plate, elutriation or any other convenient technique.
  • separation techniques include, but are not limited to, those based on differences in physical (density gradient centrifugation and counter-flow centrifugal elutriation), cell surface (lectin and antibody affinity), and vital staining properties (mitochondria-binding dye rhol23 and DNA-binding dye, Hoechst 33342).
  • Techniques providing accurate separation include but are not limited to, FACS, which can have varying degrees of sophistication, e.g., a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc.
  • the antibodies can be conjugated to identifiable agents including, but not limited to, enzymes, magnetic beads, colloidal magnetic beads, haptens, fluorochromes, metal compounds, radioactive compounds or drugs.
  • the enzymes that can be conjugated to the antibodies include, but are not limited to, alkaline phosphatase, peroxidase, urease and ⁇ - galactosidase.
  • the fluorochromes that can be conjugated to the antibodies include, but are not limited to, fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, phycoerythrin, allophycocyanins and Texas Red.
  • the metal compounds that can be conjugated to the antibodies include, but are not limited to, ferritin, colloidal gold, and particularly, colloidal superparamagnetic beads.
  • the haptens that can be conjugated to the antibodies include, but are not limited to, biotin, digoxygenin, oxazalone, and nitrophenol.
  • radioactive compounds that can be conjugated or incorporated into the antibodies are known to the art, and include but are not limited to technetium 99m ("TO, l2S I and amino acids comprising any radionuclides, including, but not limited to, l C, ⁇ and "S.
  • Any compound known in the art that can be conjugated to the antibodies by any method known in the art is suitable for use in the present invention as long as the cells selected by such method retain viability and the ability to differentiate in vi tro .
  • the method should permit the removal to a residual amount of less than about 20%, preferably less than about 5%, of the non-stem cell populations.
  • Cells may be selected based on light-scatter properties as well as their expression of various cell surface antigens.
  • the purified stem cells have low side scatter and low to medium forward scatter profiles by FACS analysis. Cytospin preparations show the enriched stem cells to have a size between mature lymphoid cells and mature granulocytes.
  • cells are initially separated by a coarse separation, followed by a fine separation, with positive selection of a marker associated with the stem cells and negative selection for markers associated with lineage committed cells.
  • RT/PCR reverse transcription coupled polymerase chain reaction
  • stromal cells including, but not limited to, mouse, porcine or human cells are provided.
  • Mouse stromal cells may come from various strains, including, but not limited to, AC3 or AC6 (otherwise referred to herein as "SySl” or “AC6.21”).
  • Stromal cells are derived from mouse, porcine or human bone marrow by selection for the ability to maintain human stem cells, and the like.
  • Stromal cells can be obtained from bone marrow, fetal thymus or fetal liver, and provide for the secretion of growth factors associated with stem and progenitor cell maintenance and differentiation.
  • the mixed stromal cell preparations may be freed of hematopoietic cells employing appropriate monoclonal antibodies for removal of the undesired cells, e.g., with antibody-toxin conjugates, antibody and complement, etc.
  • monoclonal antibodies for removal of the undesired cells, e.g., with antibody-toxin conjugates, antibody and complement, etc.
  • cloned stromal cell lines may be used where the stromal lines may be allogeneic or xenogeneic.
  • the population enriched for stem cells is preferably deposited into individual wells of a 96 well plate using an automated cell deposition unit (ACDU) on a fluorescence activated cell sorter (FACS) , although any method known in the art may be used.
  • ACDU automated cell deposition unit
  • FACS fluorescence activated cell sorter
  • the cells are CD34*Thy- 1 * LIN " .
  • These single cells are then preferably co-cultured on a mouse stromal line (AC6.21) with cytokines in amounts sufficient to induce differentiation of stem cells.
  • cytokines and concentrations are provided in the examples; however, any cytokine and concentration thereof which maintains viability and induces differentiation is suitable.
  • a suitable medium is basal media, e.g., RPMI or IMDM or a 1:1 combination supplemented with fetal calf serum (FCS) .
  • Cells can also be grown in myeloid long term culture medium (Stem Cell Technologies, Inc.) consisting 12.5% FCS, 12.5% horse serum, 1 x 10" 4 M 2- mercaptoethanol in alpha medium supplemented with 2 mM L- glutamine, 0.2 mM inositol and 20 mM folic acid.
  • Another suitable growth medium is 10% FCS, 1:1 RPMI/IMDM, 2 mM L- glutamine, 1 x 10" 4 M 2-mercaptoethanol, and 1 mM sodium pyruvate.
  • cytokines known to act on the most primitive cells will be included in the culture medium.
  • the cytokines include, but are not limited to, at least one of interleukin 3 (IL3), interleukin 6 (IL6) , granulocyte macrophage colony stimulating factor (GMCSF) , granulocyte colony stimulating factor (GCSF) , stem cell factor (SCF) , insulin-like growth factor 1 (IGF1), basic fibroblast growth factor (bFGF) , interleukin 7 (IL7), interleukin 4 (IL4) , and interleukin 11 (IL11) .
  • the cytokines include IL3, IL6, GMCSF and SCF.
  • the cytokines include IL3, IL6, GMCSF, SCF, IGF and bFGF during the first week of culture.
  • the second and third week of culture is performed in the presence of cytokines acting on cells at later stages of differentiation and will drive differentiation into more than one specific lineage.
  • Suitable cytokines are those enumerated above with the exception of IL3.
  • the cytokines are IL6, IL7, GMCSF and SCF. More preferably, the cytokines are IL6, IL7, GMCSF, SCF, IGF1 and bFGF.
  • the cells are then harvested, the RNA extracted and amplified for transcripts which encode specific markers for at least two lineages.
  • the lineage-specific transcripts are those occurring early in differentiation.
  • the lineage-specific transcripts may encode erythroid, lymphoid or myeloid specific proteins.
  • the lymphoid-specific transcripts include, but are not limited to, those encoding CD19, B220, RAG1, CD20 or RAG2.
  • the lymphoid transcript is that encoding CD19.
  • the myeloid transcripts include, but are not limited to, that encoding CD33.
  • the erythroid- specific transcripts include, but are not limited to, those encoding spectrin, GATAl and ⁇ -globin.
  • oligonucleotide primers necessary for this reaction include, but are not limited to, oligo dT primers, random primers or specific downstream primers.
  • the primer is oligo dT.
  • oligonucleotide primers Any suitable sets of oligonucleotide primers may be used.
  • the oligonucleotide primers specific to CD1 have the following sequences:
  • the oligonucleotide primers specific to CD33 have the sequences: 5'-TTACAGCCCT GCTCGCTCTTTG-3 ' ; and 5'-CAAGAATCAGCCTTTGGTCCC -3'.
  • a second, subsequent, step of analysis may be performed using a set of nested primers to CD19 cDNA sequences.
  • the nested oligonucleotide primers for the CD19 cDNA have the sequences: 5'-ATTGTCACCG TGGCAACCTGAC-3 ' ; and 5'-GGACCAGGGCTCTTTGAAGATG-3 • .
  • the nested PCR amplification is not limited to CD19 and may include any lineage specific marker.
  • the amplification of transcripts is by reverse transcription coupled polymerase chain reaction, although any method of RNA amplification or RNA coupled DNA amplification known in the art may be used. These include, but are not limited to, the self-sustained sequence replication (3SR) and branched DNA (bDNA) amplification methods.
  • 3SR self-sustained sequence replication
  • bDNA branched DNA
  • Analyses of the amplified transcripts may be performed by any method known in the art, including, but not limited to, capillary electrophoresis, agarose gel electrophoresis, fluorescence of fluorescently tagged primers, binding of biotinylated primers and high performance liquid chromatography.
  • the monitoring is by capillary electrophoresis.
  • Bone Marrows harvested from normal adults were subjected to Ficoll-Hypaque density gradient separation (1.077 g/cm 3 ) and bone marrow mononuclear cells were selected for CD34 * cells by incubating with QBEND10 anti-CD34 antibody (IOM34, AMAC, Inc.) followed by Dynabeads M450 sheep anti-mouse IgGl (Dynal Inc., Oslo, Norway). At the end of the incubations, CD34* cells were bound to the Dynabeads through the interaction between anti-CD34 antibody and sheep anti-mouse IgGl. The unbound cells were removed by pipetting while retaining the cells bound to Dynabeads by placing the tube containing the cells against a magnet.
  • QBEND10 anti-CD34 antibody IOM34, AMAC, Inc.
  • Dynabeads M450 sheep anti-mouse IgGl Dynabeads M450 sheep anti-mouse IgGl
  • CD34 * cells were released from the Dynabeads by enzymatic digestion with O-sialoglycoprotein endopeptidase (Accurate Chemical, Westbury, New York) .
  • the CD34* cells were stained with monoclonal anti-CD34 PR3-sulforhodamine, anti CD14 PR4-fluorescein, anti-CD15 PR9-Fluorescein, anti-CDw90 (Thy-1) PR13-biotin, streptavidin and biotin phycoerythrin.
  • Fluorescently stained cells were sorted for CD34*Thy-l * LIN" (LIN " is CD14 " and CD15 " ) stem cells and individual cells were deposited into wells of a 96-well plate at a concentration of one cell per well using an automated cell deposition unit on the FACS.
  • the culture medium contained 10% FCS, 2 mM gluta ine, 1 mM sodium pyruvate, 5 x 10 "5 M ⁇ -mercaptoethanol ( ⁇ -ME), IMDM/RPMI (50%/50%), 1% Penicillin/Streptomycin, interleukin-3 (IL3, Sandoz, 10 ng/ml), IL-6 (Sandoz, 10 ng/ml), insulin like growth factor-1 (IFG-1, R&D Systems, 10 ng/ml), basic fibroblast growth factor-1 (bFGF, R&D System, 10 ng/ml), stem cell factor (SCF, R&D Systems, 50 ng/ml) and granulocyte macrophage colony stimulating factor (GMCSF, 10 ng/ml) .
  • the cytokine combination was changed to IL6 (10 ng/ml), IL7 (R&D Systems, 10 ng/ml), IFG-1 (10 ng/ml), bFGF-1 (10 ng/ml), SCF (50 ng/ml) and GMCSF (10 ng/ml) .
  • IL6 10 ng/ml
  • IL7 R&D Systems
  • IFG-1 10 ng/ml
  • bFGF-1 10 ng/ml
  • SCF 50 ng/ml
  • GMCSF GMCSF
  • a 96 well culture plate was set up in a limiting dilution format ranging from 100 cells per well to 0.78 cells per well which was utilized to determine the stem cell frequency based on the cobblestone area forming cell (CAFC) morphology determination assay as described by Ploemacher et al. (1989) Blood 74:2755-2653.
  • the cells in this plate were maintained with AC6.21 and the same culture media as the single cell plates except only IL-6 and leukemia inhibitory factors LIF (10 ng/ml each) were used as cytokines.
  • This plate was scored for CAFCs after 5 weeks of culture.
  • CAFC frequency was calculated from the fraction of nonresponding cultures, using Poisson distribution.
  • the CAFC assay also allowed validation of the RT/PCR based assay for stem cell frequency in a given specimen.
  • RNA isolation and RT/PCR After 2-3 weeks of cell culture, RNA was isolated from each of the 96 wells showing cell growth. This was performed by aspirating culture media and adding 100 ⁇ L of RNA STAT60 1 " solution (Tel-Test "B” Inc, Friendswood, TX) to extract RNA from small numbers of cells. Extraction and precipitation of RNA was performed according to the protocol provided by the manufacturer. RNA was also isolated from AC6.21, KG1A and Daudi cell lines to be utilized in the control reactions. RNA obtained from each well was reverse transcribed to convert mRNA into cDNA.
  • the reverse transcription reaction was performed in a 20 ⁇ L volume containing 1 uM oligo d(T) 18 , 1 mM dNTPs, 2.5 mM MgCl 2 , 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 10 mM dithiothreitol (DTT) , 10 Units of RNA inhibitor and 100 Units of RNAse H minus M-MLV reverse transcriptase (Promega Corp., Madison, WI) .
  • the reactions were performed at 42°C for 30 minutes followed by 90°C incubation for 5 minutes. PCR reactions were performed according to the method described by Saiki et al. (1988) Science 239:487-490.
  • PCR master mix 70 ⁇ L were dispensed directly to the 20 ⁇ L RT reaction volume.
  • the final 100 ⁇ L PCR reaction contained 2 mM MgCl 2 , 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 0.5 Units of AmpliTaq DNA polymerase (Perkin Elmer Corp.), 0.25 ⁇ M each CD19 and CD33 primers.
  • the primer sequences for CD19 and CD33 are as follows:
  • CD19F3 primer 5'-ATGTGGGTAATGGAGACGGGTC-3 ' CD19B7 primer 5 ⁇ -TGGGGTCAGTCATTCGCTTTC -3' CD33F5 primer 5 • -TTACAGCCCTGCTCGCTCTTTG-3 ' CD33B13 primer 5'-CAAGAATCAGCCTTTGGTCCC -3'
  • PCR reactions were performed in a Perkin Elmer thermocycler 9600 machine by incubating the samples at 95°C, 1 min. followed by 95°C, 10 sec.; 56°C, 20 sec; 72°C, 20 sec. (35 cycles), and finally 72°C, for 5 minutes.
  • a second round of PCR was performed on all samples using a CD19 nested primer set to further amplify CD19 transcripts that are present in low copy numbers. Nested PCR reactions were performed using 2 ⁇ L of the first round reaction volume utilizing the following CD19 primer set:
  • CD19F4 primer 5'-ATTGTCACCGTGGCAACCTGAC-3 '
  • CD19B5 primer 5'-GGACCAGGGCTCTTTGAAGATG-3 '
  • Reaction conditions employed were similar to the first round PCR except 20 cycles were performed. Reaction products were analyzed by gel electrophoresis and capillary electrophoresis.
  • PCR products bv Capillary Electrophoresis
  • QlAquickTM PCR purification columns Qiagen, Inc. Chatsworth, CA
  • Samples were separated on a Biofocus 3000 capillary electrophoresis system (Bio-Rad Laboratories, Hercules, CA) using a 24 cm x 50 cm coated capillary and 0.267 M Tris-Borate buffer, pH 8.3 with polymer modifier (PCR buffer), 0.01% sodium azide.
  • FIG. 1 shows an example of a capillary electrophoresis read-out that is positive for the CD19 amplification product.

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Abstract

La présente invention concerne des méthodes de détection et de quantification de cellules souches hématopoïétiques dans une population de cellules hématopoïétiques que l'on obtient puis que l'on traite afin d'enrichir des cellules souches. On dépose des cellules simples de la population enrichie sur un substrat de croissance approprié, tel qu'un puits ménagé dans une plaque de microtitrage à 96 puits. On met ensuite en culture des dépôts de cellules simples, dans des conditions appropriées pour maintenir la viabilité et induire une différentiation. On dose ensuite les populations résultantes pour déterminer l'expression de transcrits d'ARN messager spécifiques de lignage, et notamment d'au moins deux lignages différents. On compte que la présence de transcrits spécifiques de deux lignages différents ou de davantage révèle la présence initiale d'une cellule souche.
PCT/EP1995/004480 1994-11-14 1995-11-14 Methode de mesure de cellules souches a lignage multiple et son procede d'utilisation WO1996015259A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU41167/96A AU4116796A (en) 1994-11-14 1995-11-14 Assay for the measurement of multilineage stem cells and methods of use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US33998594A 1994-11-14 1994-11-14
US08/339,985 1994-11-14

Publications (2)

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WO1996015259A2 true WO1996015259A2 (fr) 1996-05-23
WO1996015259A3 WO1996015259A3 (fr) 1996-09-19

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PCT/EP1995/004480 WO1996015259A2 (fr) 1994-11-14 1995-11-14 Methode de mesure de cellules souches a lignage multiple et son procede d'utilisation

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AU (1) AU4116796A (fr)
WO (1) WO1996015259A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005030999A1 (fr) * 2003-09-25 2005-04-07 Dana-Farber Cancer Institute, Inc Procedes permettant de detecter des cellules specifiques d'un lignage
US7045353B2 (en) 2000-08-01 2006-05-16 Yissum Research Development Company Of The Hebrew University Of Jerusalem Directed differentiation of human embryonic cells

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994008039A1 (fr) * 1992-10-02 1994-04-14 Systemix, Inc. Procede d'enrichissement de cellules souches hematopoietiques humaines a l'aide de la proteine c-kit

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7045353B2 (en) 2000-08-01 2006-05-16 Yissum Research Development Company Of The Hebrew University Of Jerusalem Directed differentiation of human embryonic cells
US7772001B2 (en) 2000-08-01 2010-08-10 Yissum Research Development Company Of The Hebrew University Of Jerusalem Directed differentiation of embryonic stem cells into an endoderm cell
WO2005030999A1 (fr) * 2003-09-25 2005-04-07 Dana-Farber Cancer Institute, Inc Procedes permettant de detecter des cellules specifiques d'un lignage

Also Published As

Publication number Publication date
WO1996015259A3 (fr) 1996-09-19
AU4116796A (en) 1996-06-06

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