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WO1996039489A1 - Isolation de cellules souches hematopoïetiques de mammaliennes - Google Patents

Isolation de cellules souches hematopoïetiques de mammaliennes Download PDF

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Publication number
WO1996039489A1
WO1996039489A1 PCT/US1996/009415 US9609415W WO9639489A1 WO 1996039489 A1 WO1996039489 A1 WO 1996039489A1 US 9609415 W US9609415 W US 9609415W WO 9639489 A1 WO9639489 A1 WO 9639489A1
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Prior art keywords
hematopoietic stem
stem cells
cells
dye
neg
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PCT/US1996/009415
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English (en)
Inventor
Margaret A. Goodell
A. Stewart Conner
Richard C. Mulligan
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Whitehead Institute For Biomedical Research
Massachusetts Institute Of Technology
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Priority to EP96919187A priority Critical patent/EP0833893A1/fr
Publication of WO1996039489A1 publication Critical patent/WO1996039489A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Hematopoietic stem cells are multipotential cells which reside in the bone marrow and replenish all adult hematopoietic lineages through the lifetime of an animal. Lack of a purified population of stem cells has hampered definitive understanding of the factors which regulate their growth and differentiation. Several schemes for the enrichment of hematopoietic stem cells from murine bone marrow have been developed, but a phenotypically homogeneous population of stem cells has not been
  • BMTs bone marrow trsnsplants
  • non-autologous BMT may also benefit from HSC purification, as transplantation of purified HSCs may ameliorate graft versus host disease (GVH) syndromes.
  • GVH graft versus host disease
  • a homogeneous stem cell population would be advantageous for hematopoietic stem cell gene therapy.
  • the present invention is based on the discovery that unusual uptake and fluorescence properties of a dye in hematopoietic stem cells (HSCs) make it possible to purify HSCs and isolate a subpopulation of HSCs that are naturally replicating in vivo .
  • HSCs hematopoietic stem cells
  • the invention relates to a method of purifying mammalian hematopoietic stem cells from a bone marrow cell population.
  • a bone marrow cell sample is obtained and combined with a fluorescent, lipophilic vital dye which is a substrate for a multiple drug resistant protein (i.e., mdr protein) under conditions appropriate for cells to take up the dye.
  • mdr protein a multiple drug resistant protein
  • substrate is defined herein as a substance which is removed by mdr. The resulting combination is exposed to an excitation
  • the amount of dye contained by each population of cells resolved with the emission wavelength is determined.
  • the population of nucleated cells which contains the smallest amount of dye at the emission wavelength is purified, mammalian HSCs.
  • a bone marrow cell sample is obtained and combined with a fluorescent, lipophilic vital dye which is a substrate for a mdr protein, under
  • the present invention also relates to a method of separating cycling HSCs from quiescent HSCs in a purified mammalian HSC sample.
  • a purified HSC sample is obtained and combined with a fluorescent, lipophilic vital dye which binds DNA and is a substrate for a mdr protein and an inhibitor of the mdr protein under
  • the amount of dye present in the purified HSCs is measured, wherein purified HSCs which contain the smallest amount of dye have a 2n quantity of DNA (i.e., which refers to the base number of chromosomes in a cell) and are designated as quiescent HSCs, and purified HSCs which contain a greater amount of dye have a greater amount of DNA (i.e. >2n) and are designated cycling HSCs. This would also apply to obtaining cycling cells from a 2n quantity of DNA (i.e., which refers to the base number of chromosomes in a cell) and are designated as quiescent HSCs, and purified HSCs which contain a greater amount of dye have a greater amount of DNA (i.e. >2n) and are designated cycling HSCs. This would also apply to obtaining cycling cells from a 2n quantity of DNA (i.e., which refers to the base number of chromosomes in a cell) and are designated as quiescent HSCs, and purified H
  • a heterogeneous population of cells such as bone marrow can be combined with the dye and the inhibitor. Cycling and non-cycling populations are determined as described above. In this case a heterogenous population of cycling cells are isolated and can be used for infecting with retroviruses. Further, the invention relates to a method of
  • identifying an agent which is a fluorescent, vital dye which is lipophilic for use in purifying mammalian HSCs In one embodiment, a bone marrow cell sample is obtained and combined with the agent under conditions appropriate for the cells to take up the agent. The resulting bone marrow cell sample is obtained and combined with the agent under conditions appropriate for the cells to take up the agent. The resulting bone marrow cell sample is obtained and combined with the agent under conditions appropriate for the cells to take up the agent. The resulting
  • the agent is a fluorescent vital dye which is lipophilic for use in purifying HSCs.
  • a bone marrow cell sample is obtained and combined with the agent under conditions appropriate for the cells to take up the agent. The resulting combination is exposed to an excitation
  • the agent is a fluorescent, vital dye which is lipophilic for use in purifying HSCs.
  • the present invention also relates to a method of transplanting bone marrow in a mammalian host, such as a human, comprising introducing into the host the purified HSCs described herein.
  • the invention further relates to a method of in vivo administration of a protein comprising infecting or transfecting a purified HSC with a construct comprising DNA or RNA which encodes a protein of interest and introducing the infected or transfected purified HSC into the host, in which the protein of interest is
  • exogenous DNA encoding a protein of interest is infected or transfected into a cycling HSC.
  • the present invention also relates to hematopoietic stem cells purified using or obtainable by (obtained by) the methods described herein.
  • the purified HSCs of the present invention are CD34 neg .
  • the purified HSCs are side population (SP) cells.
  • the purified HSCs have at least one of the following characteristics: glycophorin A neg , CD38 low/neg , CD13 neg , CD15 neg , CD19 neg and CD20 neg .
  • the present invention allows for the use of purified HSCs in bone marrow transplants.
  • the method of separating cycling HSCs from quiescent HSCs provides a means of obtaining cycling HSCs, separated from quiescent HSCs for transfection with a construct encoding a protein of interest.
  • recombinant retroviral are unable to integrate into quiescent HSCs.
  • the ability to specifically transfect cycling HSCs will provide greater success in in vivo administration of proteins to or by HSCs.
  • Figure 1A is a graph of Hoechst red versus blue fluorescence on a linear scale of whole murine normal bone marrow stained with Hoechst 33342 stain, in which the boxed region represents the hematopoietic stem cell activity, which is 0.1% of the total bone marrow cell population.
  • Figure IB is a graph illustrating the analysis of Sca- 1 and lineage marker staining of whole murine normal bone marrow.
  • Figure IC is a graph of Hoechst red versus blue fluorescence on a linear scale of whole murine normal bone marrow stained with Hoechst 33342 stain, in which the region indicated in Figure IB is used as a live gate in conjunction with the gate to exclude red and dead cells.
  • Figure ID is a graph illustrating the analysis of Sca- 1 and lineage marker staining of whole murine normal bone marrow cells which fall exclusively into the boxed region indicated in Figure 1A.
  • Figure IE is a graph of Hoechst red versus blue fluorescence on a linear scale of Sca-1 enriched murine normal bone marrow stained with Hoechst 33342 stain, in which the specific side region indicated in Table 2 is delineated.
  • Figure 2A is a graph of Hoechst red versus blue fluorescence on a linear scale of whole bone marrow stained with Hoechst 33342.
  • Figure 2B is a graph of Hoechst red versus blue fluorescence on a linear scale of whole bone marrow stained with Hoechst 33342 in the presence of Verapamil.
  • Figure 3A is a graph of Hoechst red versus blue fluorescence on a linear scale of purified HSCs restained with Hoechst 33342 in the presence of verapamil.
  • Figure 3B is a graph of propidium iodide (PI) versus cell number of purified HSC stained with PI.
  • Figure 4A is a graph of Hoechst red versus blue fluorescence on a linear scale of human whole bone marrow cells stained with Hoescht 33342 stain.
  • Figure 4B is a graph of Hoechst red versus blue fluorescence on a linear scale of human cord blood cells stained with Hoescht 33342 stain.
  • Figure 5A is a graph of Hoechst red versus blue fluorescence on a linear scale of porcine bone marrow cells stained with Hoescht 33342 stain.
  • Figure 5B is a graph of Hoechst red versus blue fluorescence on a linear scale of Rhesus bone marrow cells stained with Hoescht 33342 stain.
  • the invention relates to a method of purifying
  • HSCs mammalian hematopoietic stem cells
  • a bone marrow cell sample is obtained and combined with a fluorescent, lipophilic vital dye which is a substrate for a multiple drug resistant protein (i.e., mdr protein) under conditions appropriate for cells to take up the dye.
  • mdr protein a multiple drug resistant protein
  • substrate is defined herein as a substance which is removed from a cell by mdr. The resulting combination is exposed to an
  • excitation wavelength which results in fluorescence of the dye which is measured at an emission wavelength and the amount of dye exhibited by each population of cells is determined.
  • a bone marrow cell sample is obtained and combined with a fluorescent, lipophilic vital dye which is a substrate for a mdr protein, under
  • purifying HSCs has been used to purify side population (SP) cells from four types of mammals: murine, human, monkey and porcine. Characterization of the murine SP cells indicate they are purified HSCs. Further characterization of the human, monkey and porcine SP cells will confirm they are purified HSCs. Applicants have shown that the purified HSCs described herein successfully repopulated stem cell activity in lethally irradicated recipients, providing 1000 fold enrichment of HSC activity in mouse bone marrow. In addition, the HSCs of the present invention have been shown to allow survival of lethally irradiated recipients.
  • the present invention relates to a method for the isolation or purification of mammalian HSCs, and is based on the use of FACS analysis of bone marrow cells stained with a fluorescent vital dye. Starting with untreated bone marrow, 1,000 fold enrichment of in vivo HSC reconstitution activity can be consistently achieved. The purification strategy has also led to a method for
  • the dye which can be used in the method for purifying HSCs is a fluorescent, vital dye which is a substrate for a mdr protein.
  • the dye is also lipophilic.
  • the dye In the method of separating cycling HSCs from quiescent HSCs, the dye must also be a DNA binding dye.
  • one dye can be used in the method for purifying HSCs and in the method for separating cycling HSCs from quiescent HSCs.
  • Hoechst 33342 dye Hoechst dye
  • Rhodamine 123 might be used to practice the invention, particularly in the
  • Hoechst 33342 is a fluorescent DNA-binding dye useful for flow cytometry analysis. The dye is readily taken up by live cells, in which it binds to DNA. The quantity of Hoechst 33342 fluorescence relates to DNA content in a cell and, therefore, is an indicator of cell cycle. However, as described in Example 1, when Hoechst dye was used to examine DNA content in murine bone marrow cells by standard methods, a complex fluorescence pattern was observed. When dye fluorescence was observed simultaneously at two
  • the purified mouse HSCs are: c-kif pos , Sca-1 pos , Gr-1 neg/low , Mac-1 neg/low , CD4 neg/low , CD8 neg , B220 neg , CD5 neg , CD43 pos , CD45P° S , Mie ⁇ P 05 , AA4 ncg/low , class I MHC pos , class II MHC neg ,
  • Rhodamine 123 low Rhodamine 123 low
  • WGA Wheat germ agglutinin pos .
  • This population referred to as the side population, or SP, represents 0.1% of total bone marrow.
  • SP cells were also a sub-population (approximately 5%) of the total Sca- 1+,lin low cells in the bone marrow.
  • differentiated cells and progenitors in the unfractionated competitor bone marrow rescue recipients from the otherwise lethal irradiation. This allows the long term multi- lineage repopulating functions of the input HSC to be examined separately from short-term activities.
  • irradiated recipients resulted in survival of 50% of the recipients.
  • HSCs were purified (sorted) on the basis of low Hoechst staining, to produce SP cells as described herein.
  • the sorted or SP cells were restained with Hoechst dye in the presence of verapamil.
  • Cell-cycle analysis was performed as described in Example 1 and the number of stem cells in S-G 2 M (i.e., cycling HSCs) was found to range between 1 and 3% of the purified cells (Fig. 3A). This figure correlates well with the number shown to be in S-G 2 M by propidium iodide
  • Example 4 shows the results from the experiment.
  • the present invention also relates to a method of separating cycling HSCs from quiescent HSCs in a
  • a purified stem cell sample In this method, a purified HSC sample is combined with two substances: a fluorescent, lipophilic vital dye which binds DNA and is a substrate for a mdr protein and an inhibitor of the mdr protein. This is carried out under conditions appropriate for the cells to take up the dye and the inhibitor. The amount of dye present in the HSCs is subsequently measured or observed for differences in intensity of fluorescence. The extent to which the dye is contained by a cell is in proportion to the quantity or concentration of DNA in the cell.
  • Quiescent HSCs contain less or a lower concentration of, DNA than do cycling HSCs and, therefore, quiescent cells take up less dye than is taken up by cycling HSCs.
  • Quiescent HSCs can be distinguished from (i.e., separated from) cycling HSCs on the basis of the quantity or
  • cycling HSCs concentration of the vital dye present.
  • Those which contain a lesser quantity or concentration of dye are quiescent HSCs and those which contain a larger quantity or concentration of dye are cycling HSCs.
  • a heterogeneous population of cells such as bone marrow can be combined with the dye and the inhibitor. Cycling and non-cycling populations are determined as described above. In this case a heterogenous population of cycling cells are
  • the invention further relates to a method of
  • a bone marrow cell sample is obtained and combined with the agent under conditions appropriate for the cells to take up the agent.
  • the resulting combination is exposed to an excitation wavelength which results in fluorescent of the agent, which is measured using an emission wavelength.
  • the amount of dye contained by each population of cells resolved with the emission wavelength is determined. If distinct populations of nucleated cells are observed in which one of the population of cells contains the smallest amount of dye and the population is purified, mammalian HSCs, then the agent is a fluorescent vital dye which can be used in purifying hematopoietic stem cells.
  • an agent is identified by combining a bone marrow cell sample and an agent to be assessed, under conditions appropriate for the cells to take up the agent.
  • the resulting combination is exposed to an excitation wavelength which results in fluorescence of the dye.
  • the dye fluorescence is measured using two emission wavelengths simultaneously, so that distinct populations of live bone marrow cells are resolved on the basis of fluorescence.
  • Dye uptake is assessed in each population of cells. If distinct populations of cells are observed in which one of the population of cells contains the least amount of dye at both wavelengths and the
  • the agent is a fluorescent, vital dye which is for use in purifying HSCs.
  • the stem cell purification strategy described herein can be used with any suitable mammalian (e.g., vertebrate) species.
  • Applicants have shown that SP cells can be purified from four types of mammals: mice, humans, monkeys and pigs.
  • adult human bone marrow cells and human cord blood cells have been stained with Hoechst 33342 and a staining pattern remarkably similar to that which was observed for murine cells was observed for these cells.
  • the frequency of cells in the human side population is also approximately 0.03% (range 0.01% to 0.05%).
  • HSCs from porcine bone marrow and monkey bone marrow were purified by using the method of the present invention. Further
  • the bone marrow cells can be obtained by extracting a cell suspension from the femurs and/or tibias of the mammal, passing the cells through an orifice (e.g., an 18 gauge needle) and pelleting the cells by centrifugation.
  • an orifice e.g., an 18 gauge needle
  • the HSCs for use in the methods of the present
  • inventions can be obtained from any suitable mammalian source such as rodent (e.g., rats, mice), primate, dog, pig, cat, monkey, and/or human sources.
  • rodent e.g., rats, mice
  • primate dog, pig, cat, monkey
  • human sources e.g., human sources
  • the purified HSCs can be obtained using any suitable method for purifying HSCs.
  • the method for purifying HSCs as described herein can be used.
  • a method of purifying HSCs based on cell surface markers can be used (Visser, J.W., et al . , J. Exp. Med . , 159:1576-90 (1984); Pallavicini, M.G., et al ., Exp. Hematol . , 13:1173-81 (1985); Spangrude, G.J., et al .
  • the excitation wavelength used in the method of purifying HSCs is a suitable wavelength which will excite the particular dye chosen to a measurable extent.
  • an appropriate excitation wavelength is from about 250 nm to about 450 nm, and in a particular embodiment, is about 350 nm.
  • Hoechst dye emission can be detected at a range of wavelengths, from about 400 nm to about 700 nm, and in a particular embodiment, about 600 nm. In another embodiment about 450 nm and about 650 nm can be used simultaneously to detect Hoechst dye emission.
  • the fluorescence of the dye can be measured at one emission wavelength.
  • two emission wavelengths can be used
  • Suitable emission wavelengths are those which will measure the fluorescence of the dye chosen so that distinct
  • the fluorescence of the Hoechst dye is measured at two wavelengths using a 450 band pass (BP) 20 and a 675 edge filter long pass (EFLP) optical filter.
  • BP band pass
  • EFLP edge filter long pass
  • the fluorescence of the Hoechst dye can also be measured using only the red emission wavelength (i.e., 675 nm) to obtain purified HSCs. This was a surprising result since normally 450 nm emission wavelength is used with Hoechst dye because that is its peak emission wavelength.
  • the amount of dye used in the methods of the present invention functionally will generally be from about 1 ⁇ g/ml to about 20 ⁇ g/ml dye, preferably from about 5 ⁇ g/ml to about 15 ⁇ g/ml dye and in particular, about 5 ⁇ g/ml dye.
  • the staining time with the dye i.e., the length of time cells are exposed to dye
  • the staining time with the dye varies depending on the temperature at which staining is to occur and the dye concentration in the methods of the present invention.
  • staining can occur overnight or over a number of days at the appropriate temperature.
  • the staining time with the dye can be from about 30 minutes to about 180 minutes, preferably between about 60 minutes to about 120 minutes.
  • Hoechst dye is about 90 minutes.
  • the staining time with the Hoechst dye is about 60 minutes.
  • the temperature at which staining with the dye can be carried out is from about 4°C to about 45°C, preferably about 15°C to about 45°C, and in particular, about 37°C in the methods of the present invention.
  • the temperature at which staining with the dye can be carried out can also be room temperature (i.e., about 25°C).
  • Hoechst dye is used to stain a bone marrow cell population and, thus, the HSCs it contains, for 90 minutes at 37°C.
  • 10 ⁇ g Hoechst dye is used to stain the murine bone marrow cell population for 60 minutes at 37°C.
  • human and monkey bone marrow were stained for 120 minutes at 37°C.
  • porcine bone marrow was stained for 90 minutes at 37°C.
  • an inhibitor of the mdr protein is a substance or agent which interferes with the activity of the mdr protein in the HSCs. That is, an inhibitor of the mdr protein is a substance or agent which interferes with the ability of the mdr protein to remove the dye from the HSCs.
  • Inhibitors of the mdr protein include verapamil, antibodies directed against mdr (i.e., anti-multiple drug resistant protein antibody), reserpine, PAK-104P,
  • multiple drug resistant protein includes the multiple drug resistant (mdr) protein and proteins which exhibit mdr-like activity (i.e., an mdr-like efflux of a dye from a HSC).
  • mdr multiple drug resistant protein
  • proteins which exhibit mdr-like activity i.e., an mdr-like efflux of a dye from a HSC.
  • analogs or derivatives of the mdr protein are included in the term “multiple drug resistant protein”.
  • quiescent HSCs a low amount or quantity of DNA (i.e., 2n DNA or base number of chromosomes in a cell) in HSCs indicates the presence of quiescent HSCs (i.e., HSCs cells in the G 0 -G 1 phase) and a high amount or quantity of DNA (i.e., >2n DNA) in HSCs indicates the presence of cycling HSCs (i.e., HSCs cell in the S-G 2 M phase).
  • DNA i.e., 2n DNA or base number of chromosomes in a cell
  • cycling HSCs i.e., HSCs cell in the S-G 2 M phase
  • the HSCs of the present invention can be transplanted into a host (e.g., mammal, particularly human) in need of a bone marrow transplant (e.g., an irradiated host or a host undergoing chemotherapy).
  • a host e.g., mammal, particularly human
  • a bone marrow transplant e.g., an irradiated host or a host undergoing chemotherapy.
  • the HSCs of the present invention can be used to treat diseases or
  • the present invention further relates to a method of providing a host with purified HSCs comprising the step of introducing into the host the purified HSCs described herein.
  • a method of providing a host with purified HSCs comprising the step of introducing into the host the purified HSCs described herein.
  • the methods of the present invention can be used for in vivo administration of protein by transfecting or infecting purified HSCs with recombinant vectors or
  • HSCs comprising DNA which encodes a protein of interest.
  • cycling or quiescent purified HSCs are transfected or infected with recombinant constructs comprising DNA or RNA which encodes a protein of interest.
  • Previous attempts to infect unmanipulated HSCs with recombinant retroviral vectors have met with mixed success, presumably at least in part due to the inability of vectors to integrate into quiescent HSCs, which as shown herein constitute the majority of the HSCs population.
  • the method of separating cycling HSCs from quiescent HSCs allows for greater success in infecting HSCs with recombinant retroviral vectors.
  • the "administration of protein" by definition includes the delivery of a
  • a purified cycling HSC containing a vector, wherein the vector contains a DNA or RNA sequence which expresses a protein of interest can be administered to a host under conditions in which the protein of interest is expressed in vivo (see e.g., United States patent Number 5,399,346 which is herein incorporated by reference).
  • the ability to obtain populations of cycling stem cells also has important implications for hematopoietic stem cell therapy, including administration of the cells, as obtained by the present method as modified, such as by the introduction of an exogenous gene encoding a protein of interest (e.g., a therapeutic protein).
  • a protein of interest e.g., a therapeutic protein
  • Example 1 Characterization of Hoechst 33342 fluorescence on whole murine bone marrow.
  • Murine bone marrow was extracted from the femurs and tibias of C57B1/6 mice, a single cell suspension was made by passage of the bone marrow through an 18 gauge needle, and the cells were pelleted by centrifugation.
  • the bone marrow cells were resuspended at 10 6 cells per ml in pre- warmed DMEM contain 2% fetal calf serum, ImM HEPES, 50 units/ml Penicillin, 50 ⁇ g/ml Streptomycin, and 5 ⁇ g per ml Hoechst 33342 (Sigma) and incubated for 90 minutes at 37°C. The resolution of these populations is sensitive to the staining time and the Hoechst dye concentration (Elwart, J.W. and Dormer, P., Cytometry, 11:239-43 (1990)).
  • Hoechst dye was excited by the first Argon laser at 350nm and its fluorescence was measured at two wavelengths using a 450 band pass (BP) 20 and a 675 edge filter long pass (EFLP) optical filter (Omega Optical, Brattleboro VT).
  • BP band pass
  • EFLP edge filter long pass
  • DMSP dichroic mirror short pass
  • a 640 EFLP with 640 DMLP have also been used with an Enterprise laser (Coherent).
  • PI fluorescence was also measured through the 675 EFLP (having been excited at 350 nm).
  • Hoechst "blue” represents the 450 BP filter, the standard analysis wavelength for Hoechst 33342 DNA content analysis. Cells positive for PI were seen on the far right of the Hoechst “red” (675 EFLP) axis shown, and excluded. Both Hoechst blue and red fluorescence are shown on a linear scale. Optimal cvs off of the first laser are necessary to finely resolve the stem cell population.
  • the second Argon laser at 488 nm was used to excite standard fluorochromes (e.g., fluorescein or phycoerythrin if necessary). No cross compensation was necessary. The gating on forward and side scatter was not stringent, only erythrocytes and debris were excluded. Re-analysis of sorted populations showed purity greater than 98%.
  • the Hoechst 33342-stained murine bone marrow is placed on the flow cytometer. Since the Hoechst fluorescence is analyzed on a linear scale, optimal cvs are obtained with a relatively low sample differential, but if the cells are resuspended at a sufficiently high concentration, they may still be run at 3000-5000 cells per second. The sample can be maintained at 4°C. Initially, the voltages of Hoechst- detectors are set so that the bulk identifiable population is centered when Hoechst-Blue is displayed on the Y axis, and Hoechst-red is displayed on the X axis. Red blood cells show up in the far lower left corner as cells which indicate little or no Hoechst fluorescence.
  • the red blood cells can be thresholded out.
  • the UV beam is in the second position, so this is not possible.
  • Dead cells fluoresce with propidium iodide, and show up on the far right of the profile, as very positive on the red axis.
  • a live gate is drawn to include only live nucleated cells on the basis of the above parameters.
  • a sufficient number of data points i.e., events
  • SP small side-population
  • this region is purified by fluorescence activated cell sorting (FACs) and when used in transplantation assays, described below, this region contains all of the stem cell activity in C57B1/6 mice. The frequency of cells in this region is close to 0.1%.
  • Antibody staining was performed as follows: Hoechst- stained bone marrow was suspended in Hanks Balanced Salt Solution (HBSS) containing 2% fetal calf serum, ImM HEPES, penicillin, and streptomycin (HBSS+) at 10 8 cells per ml. The antibodies that make up the lineage cocktail were added at 1/50 to 1/100 dilutions (after being titered for this cell concentration).
  • the cocktail is comprised of the following: CD4 (GK1.5, Becton Dickenson); CD8 (53-6.7, Becton Dickenson); CD5 (53-7.3, Pharmingen); B220 (RA3-6B2, Caltag); Mac-1 (M1/70.15, Caltag); Gr-1 (RB6-8C5,
  • the mixture was incubated on ice for 10 minutes, then the bone marrow was washed once in excess HBSS+ and the cells were pelleted through a serum cushion. All washes were performed in this manner.
  • the cells were resuspended in media containing Goat anti-rat antibody conjugated to phycoerythrin (mouse-serum adsorbed, Caltag), and incubated for 10 minutes on ice. After washing, the cells were resuspended in 1/3 volume rat serum (Cappel) and 2/3 HBSS+. After 10 minutes on ice, biotinylated Sca-1 antibody (E13 161-7) was added for 10 minutes on ice.
  • the cells were stained with avidin-FITC (Becton Dickenson) for 10 minutes on ice.
  • avidin-FITC Becton Dickenson
  • Goat-anti-rate-FITC may be used to detect the lineage antibodies, and streptavadin-PE for Sca-1.
  • the bone marrow cells were resuspended in HBSS+ containing 2 ⁇ g/ml propidium iodide (PI).
  • PI propidium iodide
  • the bone marrow was sometimes magnetically pre-enriched for Sca-1 positive cells using the MACS (Miltenyi Biotec) and streptavidin microbeads. This resulted in a 5 to 10 fold enrichment, and did not affect enrichment data.
  • Cells were sorted into glass tubes containing 100% fetal calf serum. An aliquot was removed and reanalyzed to establish high purity, and cells were washed and counted prior to dilution for bone marrow transplantation.
  • the Sca/lin profile on whole bone marrow is shown in Figure IB with the region shown by us and others to contain all of the stem cell activity in the mouse indicated. This region contains between 1-5% of the cells in the bone marrow after live gating out the red blood cells and dead cells as described above. This number varies depending on which secondary conjugates are used to detect Sca-1 or the lineage cocktail.
  • the Hoechst profile is heterogeneous as shown in Fig. IC. 5-10% of the cells fall into the side population. If the region indicated in Figure 1A is used as the SOLE LIVE GATE on the whole population of murine bone marrow cells, the sca/lin profile of those cells is shown in
  • bone marrow was extracted from femurs and tibias of 10 male C57B1/6-Ly-5.1 mice (National Cancer Institute) 6-10 weeks in age and purified as described in Example 1.
  • Stem cells were purified on the basis of the combination of the Hoechst side population and Sca-1+lin low fluorescence, as described in Example 1.
  • This population was selected as follows: first, a live gate was defined using Hoechst red and blue axis to exclude dead cells and debris. After collecting 10 5 events within this live gate, the SP population is able to be clearly defined. A new live gate is established on this population, as shown boxed in Figure 1A, and the fluorescence of Sca-1 and lineage markers on SP cells was displayed.
  • Transplanted cells were given intravenously by retro- orbital injection under methoxyflurane (Pittman Moore) anesthetic. Peripheral blood was taken by retro-orbital puncture under methoxyflurane anesthesia 4 months post- transplant. All animal care was in accordance with
  • the anti-Ly-5.2 and anti-Ly-5.1 hybridomas used were 104.2.1 and A20.1.7 respectively (gifts from D. Pardoll).
  • Other anti-Ly-5.2 and anti-Ly-5.1 hybridomers are available for use (Spangrude, G.J., et al . , Science, 241 : 58-62 (1988)).
  • the results are shown in Table 1.
  • the first column displays the number of Hoechst-purified stem cells (HSC).
  • the second column displays the number of competitor cells introduced (see below).
  • N represents the number of
  • transplant recipients at the time of analysis (usually the same as the number initially transplanted) in each group.
  • the mean is the mean number of HSC-derived (Ly-5.1+) nucleated peripheral blood cells present in recipients 4 months post-transplant, and SD is the standard deviation. Enrichment is calculated as the mean percentage
  • peripheral blood was analyzed 4 months post-transplant, ensuring that most of the cells present were derived from HSC. Furthermore, the
  • the results are shown in Table 2.
  • the first column indicates the region of cells tested.
  • the second column displays the number of purified cells transplanted.
  • the third column displays the number of competitor cells introduced.
  • N represents the number of transplant
  • the mean is the mean number of nucleated peripheral blood cells derived from the fractionated bone marrow present in recipients 4 months post-transplant, and SD is the standard deviation. Enrichment is calculated as the mean % Ly-5.1 + cells in the peripheral blood per purified cell introduced, divided by the mean percentage contribution of Ly-5.2 + cells per unfractionated Ly-5.2 cell introduced. All numbers are rounded to two significant digits.
  • mice The number of purified HSCs introduced into the recipient mice are shown in the first column.
  • the number of animals transplanted in each group is shown in the second column.
  • the percentage of animals surviving at least 4 months post-transplantation is shown in the third column.
  • CFU-s 12-day spleen colony formation
  • Example 1 Whole murine bone marrow was stained for 90 minutes in 5 ⁇ g/ml Hoechst 33342 as described in Example 1 with 50 ⁇ M verapamil or without verapamil (Sigma). After staining, samples were kept on ice until flow cytometry analysis as described in Example 1.
  • hematopoietic cells suggest that p-glycoprotein is in fact expressed quite widely, and that up to 65% of bone marrow cells may express mdr (Chaudhary, P.M. and Roninson, L.B., Cell, 66:85-94 (1991); Drach, D., et al . , Blood, 80:2729-34 (1991)).
  • a purification strategy based on functional properties, such as the one described herein will likely be more powerful than a scheme based on the level of mdr cell surface expression (e.g., antibody- based).
  • Example 4 Analysis of the Cell Cycle Status of Purified Hematopoietic Stem Cells
  • Example 1 Initial purification of the HSC was as described in Example 1. After sorting to 98% purity, several thousand sorted stem cells were incubated for 60 minutes at 37°C in 10 ml DMEM containing 2% fetal calf serum, lmM HEPES, Penicillin, Streptomycin, 10 ⁇ g per ml Hoechst 33342, and 50 ⁇ M verapamil. Flow cytometry analysis was as described in Example 1. Propidium iodide stain: Stem cells purified as described above were pelleted by centrifugation and resuspended in 0.1% NaCitrate, 50 ⁇ g/ml propidium iodide. After incubation on ice for 10 minutes, the cells were analyzed by standard flow cytometry procedures using 488 nm excitation. The results are shown in Fig. 3A and Fig. 3B.
  • Example 4 shows the results from the experiment.
  • HSC second column
  • peripheral blood cells derived from the sorted cells is almost identical at 2 months and 11 months, reflecting the very long term stem cell activity of all of these populations.
  • Hematopoietic stem cells from whole human bone marrow and human umbilical cord blood were identified as described in Example 1, except as described below.
  • Human SP cells were characterized following the procedure for cell surface characterization of the mouse SP cells described in Example 1. In some samples, there are some CD34-positive cells at the TOP of the SP region, near the bulk population of cells . But the majority of cells in the SP region in several samples of human marrow that we have examined are characterized as follows: Glycophorin A neg , CD38 low , CD13 neg , CD15 neg , CD19 neg , CD20 neg and CD34 neg . The human cells run at a lower frequency, closer to 0.03-0.05% in the bone marrow.
  • Hematopoietic stem cells from porcine bone marrow and monkey bone marrow were identified as described in
  • Example 1 except as indicated below. Porcine bone marrow and Rhesus macaque monkey bone marrow were characterized following the procedure for cell surface characterization of the mouse SP cells described in Example 1. In addition, fresh bone marrow was depleted of red blood cells in the case of Rhesus bone marrow by a ficoll density gradient. Nucleated cells were counted and resuspended at 10 6
  • Hoechst 33342 dye was added to a concentration of 5 ⁇ g/ml, and the cells were incubated at 37°C for 120 minutes. Bone marrow samples were run on the flow cytometer using the set-up described for the murine bone marrow.
  • the results are shown in Figures 5A and 5B.
  • the arrows indicate the presence of cells in the same region murine stem cell activity is observed (see Figure 1A).
  • the region represents 0.05% to 0.1% of the nucleated cell population in both of these animal models.
  • the phenotype of the Rhesus monkey SP cells are as follows: CD34 neg , CD38 Iow/neg , CD4 neg , CD8 neg , Glycophorin A negative, CD61 neg , and 0066 neg .
  • the LTCIC assay is considered by many in the field to represent the best in vitro assay for HSCs. As described below, this assay was performed on hematopoietic stem cells from Rhesus monkeys. Hematopoietic stem cells from Rhesus monkeys were identified as described in Example 1. The LTCIC assays were performed as described in Sutherland, H.J., et al . , Proc . Natl . Acad . Sci . , 87:3584-3588 (1990). The results are shown in Table 5.
  • the data indicates that in the monkey, SP cells, which are CD34 neg , are highly enriched for LTICs.

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Abstract

Un procédé permet de purifier des cellules souches hématopoïétiques mammaliennes provenant d'une population de cellules de moëlle osseuse. Un autre procédé permet de séparer des cellules souches hématopoïétiques actives de celles qui sont quiescentes dans un échantillon de ces cellules purifiées mammaliennes. On décrit aussi un procédé d'identification d'un agent qui est un colorant vital fluorescent et lipophile qu'on utilise pour purifier ces cellules mammaliennes, ainsi qu'un procédé de greffe de moëlle osseuse chez un mammifère, tel qu'un être humain, procédé qui consiste à introduire chez l'hôte les cellules souches hématopoïétiques purifiées décrites. On décrit en outre un procédé d'administration in vivo d'une protéine, qui consiste à transfecter une cellule souche hématopoïétique purifiée avec un produit d'assemblage comprenant de l'ADN qui code une protéine souhaitée, puis à introduire cette cellule souche chez l'hôte où cette protéine s'exprime. On décrit aussi des cellules souches hématopoïétiques purifiées à l'aide des procédés décrits.
PCT/US1996/009415 1995-06-06 1996-06-05 Isolation de cellules souches hematopoïetiques de mammaliennes WO1996039489A1 (fr)

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EP1034288A1 (fr) * 1997-12-04 2000-09-13 Duke University Procedes d'isolation et d'utilisation des cellules hematopoietiques cd7+cd34-lin-
WO2001019379A3 (fr) * 1999-09-14 2001-05-10 Children S Medical Ct Corp The Methodes de traitement des dystrophies musculaires
US7811818B2 (en) 2001-06-22 2010-10-12 Stemcells, Inc. Human liver engrafting cells isolated from adult liver tissue
WO2014072960A3 (fr) * 2012-11-12 2014-07-24 National Centre For Biological Sciences Procédé pour identifier et isoler des cellules souches pluripotentes à l'aide de la fluorescence bleue endogène
US9132167B2 (en) 2003-05-19 2015-09-15 The Trustees Of Columbia University In The City Of New York Compositions and methods for treating and preventing heart tissue degeneration and uses thereof
US9999785B2 (en) 2014-05-30 2018-06-19 Dr. Todd Frank Ovokaitys Method and system for generation and use of activated stem cells
US10040728B2 (en) 2014-06-06 2018-08-07 Todd Frank Ovokaitys Methods and compositions for increasing the bioactivity of nutrients
US10202598B2 (en) 2014-05-30 2019-02-12 Todd Frank Ovokaitys Methods and systems for generation, use, and delivery of activated stem cells
US10384985B2 (en) 2014-06-06 2019-08-20 B.K. Consultants, Inc. Methods and compositions for increasing the yield of, and beneficial chemical composition of, certain plants
US12246037B2 (en) 2020-12-08 2025-03-11 Todd Frank Ovokaitys Methods and systems for increased production of stem cells

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M.A. GOODELL ET AL.: "ISOLATION AND FUNCTIONAL PROPERTIES OF MURINE HEMATOPOIETIC STEM CELLS THAT ARE REPLICATING IN VIVO.", THE JOURNAL OF EXPERIMENTAL MEDICINE, vol. 183, no. 4, 1 April 1996 (1996-04-01), NEW YORK, N.Y., US, pages 1797 - 1806, XP000604752 *
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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999023205A1 (fr) * 1997-10-31 1999-05-14 Hsc Research And Development Limited Partnership Cellules souches hematopoietiques
EP1034288A1 (fr) * 1997-12-04 2000-09-13 Duke University Procedes d'isolation et d'utilisation des cellules hematopoietiques cd7+cd34-lin-
EP1034288A4 (fr) * 1997-12-04 2002-11-27 Univ Duke Procedes d'isolation et d'utilisation des cellules hematopoietiques cd7+cd34-lin-
US6537807B1 (en) 1997-12-04 2003-03-25 Duke University Hematopoietic stem cells
WO2001019379A3 (fr) * 1999-09-14 2001-05-10 Children S Medical Ct Corp The Methodes de traitement des dystrophies musculaires
US7811818B2 (en) 2001-06-22 2010-10-12 Stemcells, Inc. Human liver engrafting cells isolated from adult liver tissue
US8283164B2 (en) 2001-06-22 2012-10-09 Stemcells, Inc. Liver engrafting cells, assays, and uses thereof
US9132167B2 (en) 2003-05-19 2015-09-15 The Trustees Of Columbia University In The City Of New York Compositions and methods for treating and preventing heart tissue degeneration and uses thereof
WO2014072960A3 (fr) * 2012-11-12 2014-07-24 National Centre For Biological Sciences Procédé pour identifier et isoler des cellules souches pluripotentes à l'aide de la fluorescence bleue endogène
US9933367B2 (en) 2012-11-12 2018-04-03 National Centre For Biological Sciences Method to identify and isolate pluripotent stem cells using endogenous blue fluorescence
US9999785B2 (en) 2014-05-30 2018-06-19 Dr. Todd Frank Ovokaitys Method and system for generation and use of activated stem cells
US10202598B2 (en) 2014-05-30 2019-02-12 Todd Frank Ovokaitys Methods and systems for generation, use, and delivery of activated stem cells
US10907144B2 (en) 2014-05-30 2021-02-02 Todd Frank Ovokaitys Methods and systems for generation, use, and delivery of activated stem cells
US11905510B2 (en) 2014-05-30 2024-02-20 Todd Frank Ovokaitys Methods and systems for activating cells to treat aging
US10040728B2 (en) 2014-06-06 2018-08-07 Todd Frank Ovokaitys Methods and compositions for increasing the bioactivity of nutrients
US10384985B2 (en) 2014-06-06 2019-08-20 B.K. Consultants, Inc. Methods and compositions for increasing the yield of, and beneficial chemical composition of, certain plants
US10865157B2 (en) 2014-06-06 2020-12-15 B.K. Consultants, Inc. Methods and compositions for increasing the yield of, and beneficial chemical composition of, certain plants
US12246037B2 (en) 2020-12-08 2025-03-11 Todd Frank Ovokaitys Methods and systems for increased production of stem cells

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