WO2007103763A2 - Methods for generating and using stem cell profiles - Google Patents
Methods for generating and using stem cell profiles Download PDFInfo
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- WO2007103763A2 WO2007103763A2 PCT/US2007/063123 US2007063123W WO2007103763A2 WO 2007103763 A2 WO2007103763 A2 WO 2007103763A2 US 2007063123 W US2007063123 W US 2007063123W WO 2007103763 A2 WO2007103763 A2 WO 2007103763A2
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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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5073—Stem cells
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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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5082—Supracellular entities, e.g. tissue, organisms
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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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
Definitions
- Embryonic stem cells are derived from blastocysts and arise in a very early stage of embryonic development. Embryonic stem cells can be grown in large numbers in culture but are difficult to control developmentally. In addition, their use is accompanied by perceived and unresolved ethical problems.
- Adult stem cells can be found in various tissues of the adult body. Each tissue and organ in the body originates from a small population of ASCs which is committed to differentiate into the various cell types that compose the tissue. ASCs are a likely source of continuous normal tissue replenishment, as well as, recovery in case of damage or disease, throughout the life of the organism.
- stem and progenitor cells are found in many tissues and are thought to be responsible for normal development of the organism, replenishment of normal tissues, normal functions and physiology, various hormonal cycles, certain age related developments, development of certain diseases, normal and pathological immune responses, and recovery from diseases and injuries.
- HSCs hematopoietic stem cells
- hematopoietic stem cells Various types of hematopoietic and non-hematopoietic stem and progenitor cells have been found in the human blood. Endothelial progenitor cells, mesenchymal stem cells as well as fibrocytes that can mediate tissue repair have all been reported. These cells can also be found in bone marrow in the circulation and in other organs.
- HSCs from either bone marrow, peripheral blood or cord blood are widely used for replacement of ablated bone marrow and for treatment of malignant and genetic diseases.
- Bone marrow derived hematopoietic stem cells have also been reported to maintain pluripotent potential for non-hematopoietic tissues in addition to HSC. It has recently been found that the bone marrow contains more primitive stem cells than HSCs that can differentiate into other tissues and organs. Some of these ASCs belong to a well characterized subpopulation termed mesenchymal stem cells (MSCs) that can differentiate into bone, cartilage and heart muscle cells. Other pluripotent stem cells have also been detected in bone marrow. Bone marrow derived hematopoietic stem cells have also been reported to maintain pluripotent potential for non-hematopoietic tissues.
- MSCs mesenchymal stem cells
- Such mixed populations of cells have also been isolated from cord blood, adult peripheral blood, fat tissue and other organs. Under various conditions, those mixed populations can give rise to additional tissues such as blood vessels, bone, cartilage, muscle, liver, nerve cells as well as insulin secreting Langerhans cells.
- ASCs [0005] Mixed populations of ASCs have also been identified in various other tissues. Actually, every tissue and organ in the body contains stem cells that participate in intrinsic regeneration and repair during growth, trauma and disease.
- Monocyte and macrophage like cell populations that express pluripotent potential have also been observed in the circulation of healthy adults.
- Peripheral blood endothelial progenitor cells that secrete angiogenic growth factor and form tube like structures have been reported to be derived from monocyte/macrophages.
- Non- hematopoietic MSCs and pluripotent stem cells have been reported in non-mobilized human peripheral blood and such cells were also reported to reside in the monocytes/macrophage compartment.
- stem cell populations from these various sources are being tested clinically for treatment of diseases such as ischemic heart, neural injuries, neuro-degenerative diseases, diabetes, as well as other diseases that do not currently have effective treatments.
- diseases such as ischemic heart, neural injuries, neuro-degenerative diseases, diabetes, as well as other diseases that do not currently have effective treatments.
- stem cell populations will be most effective therapeutically in particular situations. It is known that in some cases pure stem cell lines have not been effective at all.
- a stem cell profile which could include an analysis of the quality and quantity of various populations of stem and progenitor cells, which reside at any given time in a tissue, could provide a useful tool for the clinical assessment of health conditions, for prognosis of various diseases, for tailoring individual therapies and following treatment efficacy as these uses become correlated with those stem cell profiles.
- methods of determining stem cell profiles from tissues isolated from individuals are needed. Ideally the methods will provide for phenotypic and functional characterization of the various populations of stem and progenitor cells in tissue samples, such as blood samples. The methods should also provide information about the viability of the various stem and progenitor cell lineages found in a sample, hi addition, such profiles should also be associated with data corresponding to health conditions, disease prognosis, therapeutic treatment results and the like.
- a simple method for phenotypical and functional characterization of various mixed populations of stem and progenitor cells from tissue samples is disclosed, hi one aspect, the method can be used to identify the status of these cells and their functions in their native host organisms.
- the method can be applied to blood tissue and can include a step of enriching the three stem cell lineages that are commonly found in both the bone marrow, cord blood and peripheral blood: the hematopoietic, endothelial and mesenchymal stem/progenitor cells.
- the variety and population of stem cells can be identified by surface marker analysis for several defined stem cell markers, by analysis of characteristic ex vivo self renewal potential, as well as by genetic analysis such as by determining gene expression profile. Such a marker analysis can be used to obtain a functional profile of stem/progenitor populations for each cell sample under various health or clinical situations.
- a series of profiles can be generated for individuals and can be included in a database along with associated health characteristics such that the database can be used as a resource in the diagnosis of health conditions and as an aid in following patient responses to treatment, among other uses.
- the data in the profiles can also be associated with stem/progenitor cell samples in a stem cell storage and banking setting and thereby add value to the stem cell bank.
- a database of profiles for certain populations can serve for statistical, epidemiological and health evaluations.
- the present disclosure covers methods for quantifying and reporting compositions of adult stem/progenitor cell populations isolated from specific tissues. [0012] The present application also is directed to the process of quantifying the stem and progenitor cell populations and reporting the quantities to the customer.
- the tissue from which a stem cell sample is obtained can be blood, placenta, ascitic fluid, skin, kidney, liver, muscle, neural tissue or fat tissue, or their mixtures.
- the tissue is unmobilized peripheral blood.
- Each of the disclosed methods can be used to generate a revenue for a business, hi addition, access to the disclosed database or the information in the profiles could also be given for a fee in order to generate a business revenue.
- the present invention includes the identification of mixed populations of stem and progenitor cells from individuals and preferably from various individual tissues of an individual.
- Suitable body tissues include any tissues that harbor stem or progenitor cells.
- Exemplary tissues include blood, cord blood, blood buffy coat, placenta, amniotic fluid, ascitic fluid, skin, kidney, liver, muscle, neural tissue, fat, tooth pulp, and the like.
- the cell populations can include hematopoietic cells, or hematopoietic committed cell lineages including lymphoid cells, erythroid cells, myeloid cells, monocytic cells, megakaryocyte cells, and the like.
- Cell populations can also include mesenchymal cells, stromal cells, fibroblasts, endothelial cells, hematopoietic cells, hematopoietic committed cell lineages and their mixtures, for example.
- Example 1 Many identification and quantitation methods are known in the art and can be used, so long as they can be used to determine the number of isolated cell populations or mixed populations of cells.
- Examples 1 and 2 set forth exemplary methods.
- One exemplary method set forth in Example 1 involves obtaining a blood sample and, following hemolysis, subjecting the sample to analysis by fluorescent activated cell sorting (FACS) according to standard methods.
- FACS fluorescent activated cell sorting
- Cell markers for which FACS information can be obtained include CD34+, CD34+38-, CDl 05, CD90, VEGFR2, VWF, VE Cadherin, and CD31, for example.
- Other tissue samples containing stem and progenitor cells could also be used, such as cord blood and others.
- body fluid such as blood
- solid tissues such as skin, liver, muscle or other tissue biopsies
- mechanical and enzymatic methods can be used to disrupt the tissue to obtain suitable cell suspensions.
- hematopoietic stem cells can be identified as cells that contain CD 34 but that lack CD 38 markers
- mesenchymal stem cells can be identified as cells that contain CD 105 and CD90
- endothelial progenitor cells can be identified as cells that contain VEGFR2, VWF, VE Cadherin and CD31, for example.
- New markers can also be incorporated into this analysis as they are discovered.
- classifications can change with time and all changes and marker identification sets are considered to be encompassed by the present profiling methods and the profiles themselves.
- Example 2 An example of another type of analysis is set forth in Example 2.
- stem and progenitor cells from tissue samples are enriched after the tissue sample is obtained.
- the cells from the sample are distributed into suitable growth media for growing all stem and progenitor cells in the sample together.
- the enriched culture is then divided and individual samples of cells are incubated in more specialized media for growing specific types of stem or progenitor cells.
- the cells can be divided and grown in media suitable for selective growth of hematopoietic stem cells, mesenchymal stem cells, endothelial stem and progenitor cells, and hematopoietic and immune stem and progenitor cells, respectively, as described in more detail in Example 2.
- FACS analysis can be used to determine the number of cells displaying particular markers at any growth stage from any of the cultures.
- an analysis could include both a FACS analysis of cells derived directly from the tissue sample in addition to analysis of samples enriched for stem and progenitor cells from the tissue sample. Such an analysis is diagramed in Table 1 below.
- the profiling method does not necessarily result in the destruction of the entire tissue sample.
- the cell sample for the disclosed analyses can be obtained from a sample obtained for stem cell storage and can be used to provide additional information about the stored sample.
- the tissue or stem/progenitor cells can be used for a medical treatment or cryo-preserved for future medical needs or used in research, as desired.
- Samples suitable for storage or use include any of the cell populations described herein, including tissue samples, enriched samples, suspensions flow direct sample, and the like.
- the invention includes a profile listing which can include the quantities of the cells in the various stem or progenitor cell populations.
- This information can be included in one or more reports.
- a report could include any or all of the following information: 1) names of each stem/progenitor cell that can be identified in the tissue, 2) the quantity of stem/progenitor cells identified in the tissue sample, 3) the percent of each type of cell identified in the sample unit, 4) the quantities of each type of cell in a healthy population, 5) the number of cells identified that carry specific cell markers and 6) identification or quantitation of certain functions that are characteristic of the stem and progenitor cell populations, such as colony formation.
- This information can be included in one or more reports and may provide a representation of the person's stem cell profile from the tissue at the time the sample was obtained.
- the report(s) can contain information about the quantity and viability of the cells from direct issue samples and from enriched populations, as described above. Reports can also include information about the tissue and individual from which it was extracted and characteristics, such as health or disease characteristics, of the individual and the tissue or other information as desired. An exemplary report is shown in Table 2 in Example 3 below.
- the stem cell profiles can be used to evaluate health conditions of individuals.
- a series of stem cell profiles from individuals can be included in a database along with information relating to the health condition of the individuals at the time of, preceding or after the cell sample is taken. Once a sufficient amount of such data is obtained and incorporated into a database the profiles can be associated with health conditions. Once such a database is generated data from a new profile can be used to predict or diagnose the health conditions in the individuals from which they are obtained.
- Such a database could be used to predict certain diseases, for prognostic evaluation of diseases, for predicting individual therapeutic responsiveness to drugs, to follow patient responses in treatments or for other uses, as desired.
- stem cell profiles can be used to assess the potential of a person's cells for stem/progenitor- based treatments for diseases that can be treated by tissue regeneration, by protein replacement, or by coagulation factors.
- diseases include diseases associated with defective biological processes such as cardiac ischemia, osteoporosis, chronic wounds, diabetes, neural degenerative diseases, neural injuries, bone or cartilage injuries, ablated bone marrow, anemia, liver diseases, hair growth, teeth growth, retinal disease or injuries, ear diseases or injury, muscle degeneration or injury, plastic surgery.
- the treatment methods can be applied to cosmetic therapies including, filling of skin wrinkles, supporting organs, supporting surgical procedures, treating burns, and treating wounds, for example.
- Specific treatment methods can include autologous or allogeneic therapies.
- the stem cell profile can be used to assess the potential of using the person's cells for preparing a therapeutic agent containing a product secreted from the aforementioned cell populations.
- the method can be accomplished by preparing a cell population by any of the methods described previously and incubating the cells in culture media for a period of time sufficient to generate secreted products.
- the secreted products can then be isolated from the culture media by known methods which one of skill in the art can appreciate will depend upon the nature of the product.
- the stem cell profile can be used to asses the potential of using the person's cells for methods of using the disclosed cell populations in gene therapy. Such methods can be accomplished by preparing cell populations by methods as described above. The cells can then be transfected with a recombinant DNA or other methods of gene manipulations and the modified cells can be introduced into a patient in need thereof.
- the disclosed methods can be used to generate revenues.
- the method can utilize any or all of the above disclosed methods including any or all of the information gathering methods, the data gathered and the profiles themselves or any other disclosed product or service.
- the information obtained and the profiles can be stored in a database together or separately.
- a fee can be charged for carrying out any of the disclosed methods on behalf of a third party such as a patient, or for providing some or all of the gathered information to a researcher, healthcare worker, health or life insurance company, provider of medical services, government body or any other entity that is interested enough in the information to pay for it, such that a business can generate revenue.
- the information can be sold for the purpose of research, for development of medical treatments and diagnostics or for any other desired use.
- This example demonstrates a method for determining a profile of stem and progenitor cells using minimal manipulation of the cells from blood.
- a sample of venous blood of about 10 to about 20 ml can be obtained aseptically in a heparinized tube.
- the sample can be withdrawn from a unit of blood or obtained directly from an individual.
- the sample is then analyzed using fluorescent activated cell sorting techniques
- Cell markers for which FACS information can be obtained include CD34+, CD34+38-, CD105, CD90, VEGFR2, VWF, VE Cadherin, and CD31, CD3, CD4, CD8, CD14, CD45 for example
- This example demonstrates a method for determining a profile of stem and progenitor cells from a blood sample.
- a sample of venous blood of about 20 to about 50 ml can be obtained aseptically in a heparinized tube.
- the sample can be obtained from a unit of blood that was withdrawn for storage or directly from an individual.
- the blood sample was then subjected to ficoll paque density gradient separation, using standard methods, to isolate mononuclear cells (MNC).
- MNC mononuclear cells
- the enriched mononuclear cells are then divided into four aliquots.
- One aliquot is plated for direct hematopoietic colony formation in semisolid media, supplemented with hematopoietic growth factors.
- About 2 x 10 5 cells per ml, are plated in semi-solid medium (methyl cellulose) supplemented with 10%FCS and growth factors that stimulate hematopoietic cell growth including, for example SCF, G-CSF, GM-CSF, IL-3, IL-6, EPO and their combinations.
- a colony count can then be determined after about two weeks.
- different colonies, distinguished by morphological criteria as are known can be counted for various progenitor lineages responsive to specific growth factors or combinations of growth factors.
- Culture conditions for growing colonies include for example, plating 1-lOxlO 6 mononuclear cells in tissue culture media such as RPMI, methyl cellulose, antibiotics, serum such as fetal calf serum, growth factors as mentioned above and incubation for example for 1- 2 weeks in a humidified 5%CO2 incubator.
- tissue culture media such as RPMI, methyl cellulose, antibiotics, serum such as fetal calf serum, growth factors as mentioned above and incubation for example for 1- 2 weeks in a humidified 5%CO2 incubator.
- One aliquot of the enriched mononuclear cell culture is plated in tissue culture media for enrichment of endothelial progenitor cells.
- Enriched mononuclear cells can be plated at about 4 x 10 5 cells per 2 ml in fibronectin precoated culture plates.
- the media used can be EBM2 supplemented with pre-selected FCS (20%) and growth factors including growth factors that stimulate endothelial progenitor cell growth, such as VEGF, bFGF, IGFl about 2-20 nanogram per ml each and their combinations.
- VEGF vascular endothelial progenitor cell growth
- the cells can be collected with trypsin-EDTA and plated at 2 x 10 5 cells per 2 ml. After an additional 6-8 days the cells can be collected and separated into two fractions, one fraction to be analyzed by FACS for endothelial cell markers such as VEGFR-2, VWF, VE-cadherin, CD31, one fraction to be analyzed using a colony formation assay for each growth factor or combination to be studied, in a known method for endothelial progenitor colony formation assay(CFU-E).
- FACS endothelial cell markers
- CD31 a colony formation assay for each growth factor or combination to be studied
- One aliquot of the enriched mononuclear cells culture can be plated in tissue culture media for enrichment of mesenchymal stem cells.
- Mononuclear cells can be plated at a density of about 4 x 10 5 cells per 2 ml in tissue culture plates in tissue culture media such as low glucose MEM supplemented with pre-selected FCS (10-20%) and growth factors that stimulate mesenchymal stem cells such as: , bFGF, M-CSF, IL6, LIF, about 20-50 nanogram per ml each, , After about 3-4 days, the nonadherent cells can be removed and the adherent cells further incubated in the same media. Media is replaced about every 3-4 days.
- the cells are collected with trypsin-EDTA and plated at about 2 x 10 5 per 2 mis. After an additional 6-8 days the cells are collected and separated into two fractions which are analyzed by FACS analysis for markers such as CD 105 and CD90 and in a colony formation assay (CFU-M).
- CFU-M colony formation assay
- tissue culture media for enrichment of the hematopoietic and immune stem/progenitor cell lineages.
- the enriched mononuclear cell culture can be plated at a density of 1 x 10 5 cells per 2ml in RPMI culture media supplemented with pre-selected FCS (10%) and growth factors combination such as: IL-6, SCF, Flt3, TPO, each factor about 50 nanogram per ml.
- Fresh media can be added about every 3 to 4 days.
- Example 3 The following example demonstrates the use of the results obtained in Examples 1 and 2 that can be organized into a concise report of a stem cell profile obtained from a tissue sample.
- the procedure set forth in Example 1 can be used to obtain data which can be used to fill each of the three columns across the first line of Table 2 for data obtained from the tissue sample that was analyzed prior to ex vivo enrichment.
- Example 2 The procedure set forth in Example 2 can be used to obtain data which can be used to fill each of the three columns across the second and third line of Table 2 for data obtained from the tissue sample that was analyzed after ex vivo enrichment.
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Abstract
A method for phenotypical and functional characterization of various populations of stem and progenitor cells from cell samples, such as tissue samples is disclosed. In one aspect, the method can be used to identify the status of these cells and their functions in individuals. The method, when applied to blood tissue, can include a step of enriching stem cell lineages that are commonly found in both the bone marrow and peripheral blood: the hematopoietic, endothelial and mesenchymal stem/progenitor cells. The variety and population of stem cells can be identified by analysis of cell markers for several defined stem cell and lineage committed markers and by analysis of characteristic ex vivo self renewal potential. Such techniques can be used to obtain a functional profile of stem/progenitor populations for each cell sample, and for various clinical conditions. The profile can also be associated with and add value to the storage and banking business of stem cells. Profiles and consecutive profiles for individuals can be included in a database along with health characteristics of the individuals. The database can be used for patient follow-up or scientific reference. Database profiles for certain populations may serve for statistical, epidemiological and health evaluations.
Description
TITLE METHODS FOR GENERATING AND USING STEM CELL PROFILES
BACKGROUND
[0001] Stem cells can be grouped into two cell types, namely embryonic stem cells (ES) and adult stem cells (ASCs). Embryonic stem cells are derived from blastocysts and arise in a very early stage of embryonic development. Embryonic stem cells can be grown in large numbers in culture but are difficult to control developmentally. In addition, their use is accompanied by perceived and unresolved ethical problems. Adult stem cells can be found in various tissues of the adult body. Each tissue and organ in the body originates from a small population of ASCs which is committed to differentiate into the various cell types that compose the tissue. ASCs are a likely source of continuous normal tissue replenishment, as well as, recovery in case of damage or disease, throughout the life of the organism. Various populations of stem and progenitor cells are found in many tissues and are thought to be responsible for normal development of the organism, replenishment of normal tissues, normal functions and physiology, various hormonal cycles, certain age related developments, development of certain diseases, normal and pathological immune responses, and recovery from diseases and injuries.
[0002] One tissue that contains ASCs, and perhaps the most widely studied tissue in animals, is blood. Most, if not all, blood cells, including red blood cells, lymphocytes, monocytes, polymorphs, and platelets originate from a specific type of ASC known as hematopoietic stem cells (HSCs). Various types of hematopoietic and non-hematopoietic stem and progenitor cells have been found in the human blood. Endothelial progenitor cells, mesenchymal stem cells as well as fibrocytes that can mediate tissue repair have all been reported. These cells can also be found in bone marrow in the circulation and in other organs.
[0003] HSCs from either bone marrow, peripheral blood or cord blood, are widely used for replacement of ablated bone marrow and for treatment of malignant and genetic diseases. Bone marrow derived hematopoietic stem cells have also been reported to maintain pluripotent potential for non-hematopoietic tissues in addition to HSC. It has recently been found that the bone marrow contains more primitive stem
cells than HSCs that can differentiate into other tissues and organs. Some of these ASCs belong to a well characterized subpopulation termed mesenchymal stem cells (MSCs) that can differentiate into bone, cartilage and heart muscle cells. Other pluripotent stem cells have also been detected in bone marrow. Bone marrow derived hematopoietic stem cells have also been reported to maintain pluripotent potential for non-hematopoietic tissues.
[0004] Such mixed populations of cells have also been isolated from cord blood, adult peripheral blood, fat tissue and other organs. Under various conditions, those mixed populations can give rise to additional tissues such as blood vessels, bone, cartilage, muscle, liver, nerve cells as well as insulin secreting Langerhans cells.
[0005] Mixed populations of ASCs have also been identified in various other tissues. Actually, every tissue and organ in the body contains stem cells that participate in intrinsic regeneration and repair during growth, trauma and disease.
[0006] Monocyte and macrophage like cell populations that express pluripotent potential have also been observed in the circulation of healthy adults. Peripheral blood endothelial progenitor cells that secrete angiogenic growth factor and form tube like structures, have been reported to be derived from monocyte/macrophages. Non- hematopoietic MSCs and pluripotent stem cells have been reported in non-mobilized human peripheral blood and such cells were also reported to reside in the monocytes/macrophage compartment.
[0007] Thus, it is clear that complex mixtures of stem and progenitor cells exist in some or all body tissues, hi addition to their well established use in bone marrow transplantation, certain stem cell populations from these various sources are being tested clinically for treatment of diseases such as ischemic heart, neural injuries, neuro-degenerative diseases, diabetes, as well as other diseases that do not currently have effective treatments. However, it remains unclear which stem cell populations will be most effective therapeutically in particular situations. It is known that in some cases pure stem cell lines have not been effective at all.
[0008] A stem cell profile which could include an analysis of the quality and quantity of various populations of stem and progenitor cells, which reside at any given time in a tissue, could provide a useful tool for the clinical assessment of health conditions, for prognosis of various diseases, for tailoring individual therapies and
following treatment efficacy as these uses become correlated with those stem cell profiles. Thus, methods of determining stem cell profiles from tissues isolated from individuals are needed. Ideally the methods will provide for phenotypic and functional characterization of the various populations of stem and progenitor cells in tissue samples, such as blood samples. The methods should also provide information about the viability of the various stem and progenitor cell lineages found in a sample, hi addition, such profiles should also be associated with data corresponding to health conditions, disease prognosis, therapeutic treatment results and the like.
SUMMARY
[0009] A simple method for phenotypical and functional characterization of various mixed populations of stem and progenitor cells from tissue samples is disclosed, hi one aspect, the method can be used to identify the status of these cells and their functions in their native host organisms. The method can be applied to blood tissue and can include a step of enriching the three stem cell lineages that are commonly found in both the bone marrow, cord blood and peripheral blood: the hematopoietic, endothelial and mesenchymal stem/progenitor cells.
[0010] The variety and population of stem cells can be identified by surface marker analysis for several defined stem cell markers, by analysis of characteristic ex vivo self renewal potential, as well as by genetic analysis such as by determining gene expression profile. Such a marker analysis can be used to obtain a functional profile of stem/progenitor populations for each cell sample under various health or clinical situations. A series of profiles can be generated for individuals and can be included in a database along with associated health characteristics such that the database can be used as a resource in the diagnosis of health conditions and as an aid in following patient responses to treatment, among other uses. The data in the profiles can also be associated with stem/progenitor cell samples in a stem cell storage and banking setting and thereby add value to the stem cell bank. A database of profiles for certain populations can serve for statistical, epidemiological and health evaluations.
[0011] The present disclosure covers methods for quantifying and reporting compositions of adult stem/progenitor cell populations isolated from specific tissues.
[0012] The present application also is directed to the process of quantifying the stem and progenitor cell populations and reporting the quantities to the customer.
[0013] Li particular embodiments, the tissue from which a stem cell sample is obtained can be blood, placenta, ascitic fluid, skin, kidney, liver, muscle, neural tissue or fat tissue, or their mixtures. Preferably, the tissue is unmobilized peripheral blood.
[0014] Each of the disclosed methods can be used to generate a revenue for a business, hi addition, access to the disclosed database or the information in the profiles could also be given for a fee in order to generate a business revenue.
[0015] Additional features and advantages are described herein, and will be apparent from, the following Detailed Description.
DETAILED DESCRIPTION
[0016] In one aspect, the present invention includes the identification of mixed populations of stem and progenitor cells from individuals and preferably from various individual tissues of an individual. Suitable body tissues include any tissues that harbor stem or progenitor cells. Exemplary tissues include blood, cord blood, blood buffy coat, placenta, amniotic fluid, ascitic fluid, skin, kidney, liver, muscle, neural tissue, fat, tooth pulp, and the like.
[0017] Any cell population containing stem and progenitor cells can be analyzed, hi an embodiment, the cell populations can include hematopoietic cells, or hematopoietic committed cell lineages including lymphoid cells, erythroid cells, myeloid cells, monocytic cells, megakaryocyte cells, and the like. Cell populations can also include mesenchymal cells, stromal cells, fibroblasts, endothelial cells, hematopoietic cells, hematopoietic committed cell lineages and their mixtures, for example.
[0018] Many identification and quantitation methods are known in the art and can be used, so long as they can be used to determine the number of isolated cell populations or mixed populations of cells. Examples 1 and 2 set forth exemplary methods. One exemplary method set forth in Example 1 involves obtaining a blood sample and, following hemolysis, subjecting the sample to analysis by fluorescent activated cell sorting (FACS) according to standard methods. Cell markers for which FACS information can be obtained include CD34+, CD34+38-, CDl 05, CD90,
VEGFR2, VWF, VE Cadherin, and CD31, for example. Other tissue samples containing stem and progenitor cells could also be used, such as cord blood and others. It is technically simpler to obtain body fluid, such as blood; but obtaining solid tissues such as skin, liver, muscle or other tissue biopsies is also within the skill of a skilled artisan, and in such case, mechanical and enzymatic methods can be used to disrupt the tissue to obtain suitable cell suspensions.
[0019] Any suitable method for identifying cell populations can be used. For example, hematopoietic stem cells can be identified as cells that contain CD 34 but that lack CD 38 markers, mesenchymal stem cells can be identified as cells that contain CD 105 and CD90 and endothelial progenitor cells can be identified as cells that contain VEGFR2, VWF, VE Cadherin and CD31, for example. New markers can also be incorporated into this analysis as they are discovered. In addition, classifications can change with time and all changes and marker identification sets are considered to be encompassed by the present profiling methods and the profiles themselves.
[0020] An example of another type of analysis is set forth in Example 2. Generally, in this method stem and progenitor cells from tissue samples are enriched after the tissue sample is obtained. To accomplish this, the cells from the sample are distributed into suitable growth media for growing all stem and progenitor cells in the sample together. The enriched culture is then divided and individual samples of cells are incubated in more specialized media for growing specific types of stem or progenitor cells. For example, with mononuclear cells enriched samples from blood, the cells can be divided and grown in media suitable for selective growth of hematopoietic stem cells, mesenchymal stem cells, endothelial stem and progenitor cells, and hematopoietic and immune stem and progenitor cells, respectively, as described in more detail in Example 2. Once the enriched individual cultures are obtained, FACS analysis can be used to determine the number of cells displaying particular markers at any growth stage from any of the cultures.
[0021] The analyses described above are not exclusive. Thus, in an embodiment, an analysis could include both a FACS analysis of cells derived directly from the tissue sample in addition to analysis of samples enriched for stem and
progenitor cells from the tissue sample. Such an analysis is diagramed in Table 1 below.
Table 1
Blood sample
4/5 sample 1/5 sample Ficoll: MNC FACS
1/4 sample I 1/4 sample I 1/4 sample 1/4 sample HSC CFU I MSC culture I ESC culture HSC culture
I FACS I FACS FACS
+ + +
I CFU-F I CFU-E CFU-HSC
[0022] The profiling method does not necessarily result in the destruction of the entire tissue sample. Instead, the cell sample for the disclosed analyses can be obtained from a sample obtained for stem cell storage and can be used to provide additional information about the stored sample. Thus, in addition to the analysis, the tissue or stem/progenitor cells can be used for a medical treatment or cryo-preserved for future medical needs or used in research, as desired. Samples suitable for storage or use include any of the cell populations described herein, including tissue samples, enriched samples, suspensions flow direct sample, and the like.
[0023] In an aspect, the invention includes a profile listing which can include the quantities of the cells in the various stem or progenitor cell populations. This information can be included in one or more reports. For example, a report could include any or all of the following information: 1) names of each stem/progenitor cell that can be identified in the tissue, 2) the quantity of stem/progenitor cells identified in the tissue sample, 3) the percent of each type of cell identified in the sample unit, 4) the quantities of each type of cell in a healthy population, 5) the number of cells identified that carry specific cell markers and 6) identification or quantitation of
certain functions that are characteristic of the stem and progenitor cell populations, such as colony formation. This information can be included in one or more reports and may provide a representation of the person's stem cell profile from the tissue at the time the sample was obtained. The report(s) can contain information about the quantity and viability of the cells from direct issue samples and from enriched populations, as described above. Reports can also include information about the tissue and individual from which it was extracted and characteristics, such as health or disease characteristics, of the individual and the tissue or other information as desired. An exemplary report is shown in Table 2 in Example 3 below.
[0024] The stem cell profiles can be used to evaluate health conditions of individuals. Thus, a series of stem cell profiles from individuals can be included in a database along with information relating to the health condition of the individuals at the time of, preceding or after the cell sample is taken. Once a sufficient amount of such data is obtained and incorporated into a database the profiles can be associated with health conditions. Once such a database is generated data from a new profile can be used to predict or diagnose the health conditions in the individuals from which they are obtained. Such a database could be used to predict certain diseases, for prognostic evaluation of diseases, for predicting individual therapeutic responsiveness to drugs, to follow patient responses in treatments or for other uses, as desired.
[0025] The present disclosure also specifically contemplates that stem cell profiles can be used to assess the potential of a person's cells for stem/progenitor- based treatments for diseases that can be treated by tissue regeneration, by protein replacement, or by coagulation factors. Such diseases include diseases associated with defective biological processes such as cardiac ischemia, osteoporosis, chronic wounds, diabetes, neural degenerative diseases, neural injuries, bone or cartilage injuries, ablated bone marrow, anemia, liver diseases, hair growth, teeth growth, retinal disease or injuries, ear diseases or injury, muscle degeneration or injury, plastic surgery. In addition, the treatment methods can be applied to cosmetic therapies including, filling of skin wrinkles, supporting organs, supporting surgical procedures, treating burns, and treating wounds, for example. Specific treatment methods can include autologous or allogeneic therapies.
[0026] The present disclosure also contemplates that the stem cell profile can be used to assess the potential of using the person's cells for preparing a therapeutic agent containing a product secreted from the aforementioned cell populations. To this end, the method can be accomplished by preparing a cell population by any of the methods described previously and incubating the cells in culture media for a period of time sufficient to generate secreted products. The secreted products can then be isolated from the culture media by known methods which one of skill in the art can appreciate will depend upon the nature of the product.
[0027] The present disclosure also contemplates that the stem cell profile can be used to asses the potential of using the person's cells for methods of using the disclosed cell populations in gene therapy. Such methods can be accomplished by preparing cell populations by methods as described above. The cells can then be transfected with a recombinant DNA or other methods of gene manipulations and the modified cells can be introduced into a patient in need thereof.
[0028] hi one aspect of the invention, the disclosed methods can be used to generate revenues. The method can utilize any or all of the above disclosed methods including any or all of the information gathering methods, the data gathered and the profiles themselves or any other disclosed product or service. In addition, the information obtained and the profiles can be stored in a database together or separately. A fee can be charged for carrying out any of the disclosed methods on behalf of a third party such as a patient, or for providing some or all of the gathered information to a researcher, healthcare worker, health or life insurance company, provider of medical services, government body or any other entity that is interested enough in the information to pay for it, such that a business can generate revenue. For example, the information can be sold for the purpose of research, for development of medical treatments and diagnostics or for any other desired use.
Example 1
[0029] This example demonstrates a method for determining a profile of stem and progenitor cells using minimal manipulation of the cells from blood. A sample of venous blood of about 10 to about 20 ml can be obtained aseptically in a heparinized tube. The sample can be withdrawn from a unit of blood or obtained directly from an
individual. The sample is then analyzed using fluorescent activated cell sorting techniques Cell markers for which FACS information can be obtained include CD34+, CD34+38-, CD105, CD90, VEGFR2, VWF, VE Cadherin, and CD31, CD3, CD4, CD8, CD14, CD45 for example
Example 2
[0030] This example demonstrates a method for determining a profile of stem and progenitor cells from a blood sample. A sample of venous blood of about 20 to about 50 ml can be obtained aseptically in a heparinized tube. The sample can be obtained from a unit of blood that was withdrawn for storage or directly from an individual. The blood sample was then subjected to ficoll paque density gradient separation, using standard methods, to isolate mononuclear cells (MNC). The enriched mononuclear cells are then divided into four aliquots.
[0031] One aliquot is plated for direct hematopoietic colony formation in semisolid media, supplemented with hematopoietic growth factors. About 2 x 105 cells per ml, are plated in semi-solid medium (methyl cellulose) supplemented with 10%FCS and growth factors that stimulate hematopoietic cell growth including, for example SCF, G-CSF, GM-CSF, IL-3, IL-6, EPO and their combinations. A colony count can then be determined after about two weeks. Alternatively, different colonies, distinguished by morphological criteria as are known can be counted for various progenitor lineages responsive to specific growth factors or combinations of growth factors. Such known morphological characteristics, which are found in the literature, are used to distinguish between various hematopoietic cell lineages. Culture conditions for growing colonies include for example, plating 1-lOxlO6 mononuclear cells in tissue culture media such as RPMI, methyl cellulose, antibiotics, serum such as fetal calf serum, growth factors as mentioned above and incubation for example for 1- 2 weeks in a humidified 5%CO2 incubator.
[0032] One aliquot of the enriched mononuclear cell culture is plated in tissue culture media for enrichment of endothelial progenitor cells. Enriched mononuclear cells can be plated at about 4 x 105 cells per 2 ml in fibronectin precoated culture plates. The media used can be EBM2 supplemented with pre-selected FCS (20%) and growth factors including growth factors that stimulate endothelial progenitor cell
growth, such as VEGF, bFGF, IGFl about 2-20 nanogram per ml each and their combinations. After about 3-4 days the non-adherent cells can be removed and the adherent cells further incubated in the same media. Media is replaced about every 3-4 days. After 6-8 days, the cells can be collected with trypsin-EDTA and plated at 2 x 105 cells per 2 ml. After an additional 6-8 days the cells can be collected and separated into two fractions, one fraction to be analyzed by FACS for endothelial cell markers such as VEGFR-2, VWF, VE-cadherin, CD31, one fraction to be analyzed using a colony formation assay for each growth factor or combination to be studied, in a known method for endothelial progenitor colony formation assay(CFU-E).
[0033] One aliquot of the enriched mononuclear cells culture can be plated in tissue culture media for enrichment of mesenchymal stem cells. Mononuclear cells can be plated at a density of about 4 x 105 cells per 2 ml in tissue culture plates in tissue culture media such as low glucose MEM supplemented with pre-selected FCS (10-20%) and growth factors that stimulate mesenchymal stem cells such as: , bFGF, M-CSF, IL6, LIF, about 20-50 nanogram per ml each, , After about 3-4 days, the nonadherent cells can be removed and the adherent cells further incubated in the same media. Media is replaced about every 3-4 days. After about 6-8 days, the cells are collected with trypsin-EDTA and plated at about 2 x 105 per 2 mis. After an additional 6-8 days the cells are collected and separated into two fractions which are analyzed by FACS analysis for markers such as CD 105 and CD90 and in a colony formation assay (CFU-M).
[0034] One aliquot is plated in tissue culture media for enrichment of the hematopoietic and immune stem/progenitor cell lineages. To this end the enriched mononuclear cell culture can be plated at a density of 1 x 105 cells per 2ml in RPMI culture media supplemented with pre-selected FCS (10%) and growth factors combination such as: IL-6, SCF, Flt3, TPO, each factor about 50 nanogram per ml. Fresh media can be added about every 3 to 4 days. After about 10 to 14days, the cells can be collected and separated into two fractions for analysis by FACS for markers such as CD45, CD34, CD133, CD3, CD14, CD56, CD4, CD8 and in a colony formation assay (CFU-E, BFU-E, CFU-GM, CFU-GEMM), using standard morphological criteria that distinguish such colonies. Example 3
[0035] The following example demonstrates the use of the results obtained in Examples 1 and 2 that can be organized into a concise report of a stem cell profile obtained from a tissue sample. The procedure set forth in Example 1 can be used to obtain data which can be used to fill each of the three columns across the first line of Table 2 for data obtained from the tissue sample that was analyzed prior to ex vivo enrichment.
[0036] The procedure set forth in Example 2 can be used to obtain data which can be used to fill each of the three columns across the second and third line of Table 2 for data obtained from the tissue sample that was analyzed after ex vivo enrichment.
Table 2
[0037] It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. It is therefore intended that such changes and modifications be covered by the appended claims.
Claims
1. A method for determining a profile of stem and progenitor cells comprising; obtaining a tissue sample from an individual, determining the cell markers that are resident on the cells from the tissue, classifying the stem and progenitor cells, identifying the number of cells in each stem and progenitor cell type, and incorporating the information into a stem and progenitor cell profile.
2. The method for determining a profile of Claim 1, wherein the cell markers are determined by fluorescence activated cell sorting analysis.
3. The method for determining a profile of Claim 1, wherein the cell markers are determined by fluorescence activated cell sorting analysis using a cell marker selected from the group of cell markers consisting of CD34+, CD34+38-, CD45, CD 105, CD90, VEGFR2, VWF, VE-Cadherin, and CD31.
4. The method for determining a profile of Claim 1, further comprising classifying cells having CD 34 and lacking CD 38 markers as Hematopoietic Stem Cells.
5. The method for determining a profile of Claim 1, further comprising classifying cells having CD 105 and CD90 as Mesenchymal Stem Cells.
6. The method for determining a profile of Claim 1, further comprising classifying cells having VEGFR2, VWF, VE Cadherin and CD31 as Endothelial Progenitor Cells.
7. A method for determining a profile of stem and progenitor cells comprising, obtaining a tissue sample from an individual, enriching the sample with stem and progenitor cells, dividing the enriched sample, selectively growing a defined stem cell or progenitor cell line from a divided portion of the enriched sample, counting the grown colonies of the defined stem cell or progenitor cell line and incorporating the information into a stem and progenitor cell profile.
8. The method of Claim 7, further comprising determining the cell markers that are resident on the enriched cells from the tissue.
9. The method of any of Claims 1-8, wherein the tissue includes a tissue selected from the group of tissues consisting of blood, umbilical cord blood, blood buffy coat, placenta, amniotic fluid, ascitic fluid, skin, kidney, liver, muscle, neural tissue, fat and tooth pulp.
10. The methods of any of Claims 1-9, wherein the stem or progenitor cells are selected from the group of cells consisting of hematopoietic cells, hematopoietic committed cells.
11. The methods of any of Claims 1 -9, wherein the stem or progenitor cells are selected from the group of cells consisting of lymphoid cells, erythroid cells, myeloid cells, monocytic cells, megakaryocytic cells, mesenchymal cells, stromal cells, fibroblasts, endothelial cells hematopoietic cells and hematopoietic committed cell lineages.
12. The methods of any of Claims 1-9, wherein the tissue includes blood and the enriched sample is enriched for mononuclear cells.
13. A method for determining a profile of stem and progenitor cells comprising, obtaining a tissue sample from an individual and dividing the sample into at least two portions, determining the cell markers that are resident on the cells from the tissue of a first portion of the tissue, classifying the stem and progenitor cells, identifying the number of cells in each stem and progenitor cell type, and incorporating the information obtained from the first portion into a stem and progenitor cell profile; enriching a second portion of the sample with stem and progenitor cells, dividing the enriched sample, selectively growing a defined stem cell or progenitor cell line from a portion of the enriched sample, counting the grown colonies of the defined stem cell or progenitor cell line and incorporating the information into the stem and progenitor cell profile.
14. A stem cell and progenitor cell profile comprising: the names of each stem/progenitor cell that can be identified in a cell sample, the quantity of stem/progenitor cells identified in the sample, the percent of each type of cell identified in the sample, and the number of cells identified that carry specific cell markers wherein this information can be included in at least one report.
15. The stem cell and progenitor cell profile of Claim 14, further comprising information in a report about the viability of the cells identified in the sample.
16. A stem cell bank comprising a series of stem and progenitor cell samples obtained from a series of individuals, wherein samples are associated with stem and progenitor cell profiles obtained from the same individuals.
17. The stem cell bank of Claim 16, wherein the stem and progenitor cell profiles are derived from the stem and progenitor cell sample stored in the library.
18. A database comprising stem and progenitor cell profiles and health conditions in association such that the health conditions are derived from individuals who originated the stem and progenitor cell profiles.
19. A method for evaluating the health of an individual comprising: obtaining a stem and progenitor cell profile from an individual, comparing the profile to a database comprising information from a series of stem cell and progenitor cell profiles correlated with the health conditions associated with the profiles, identifying health conditions associated with specific stem and progenitor cell profiles.
20. The method of Claim 19, wherein the health condition is selected from the group of health conditions consisting of a disease, a disease prognosis and a prognostic evaluation of a response to a drug.
21. The method of Claim 19, wherein the health condition is the prognostic evaluation of a response to a stem cell treatment.
22. The method of Claim 19, wherein the health condition is the prognostic evaluation of a response to a progenitor cell treatment.
23. The method of Claim 19, wherein the health condition is a disease selected from the group of diseases consisting of cardiac ischemia, osteoporosis, chronic wounds, diabetes, neural degenerative diseases, neural injuries, bone or cartilage injuries, ablated bone marrow, anemia, liver disease, allopecia, tooth growth, retinal disease or injuries, ear diseases or injury, muscle degeneration, muscle injury, perceived cosmetic deficiencies, organ support, bums and wounds.
24. The method of Claim 19, wherein the treatment is an autologous treatment and the health condition is a disease selected from the group of diseases consisting of cardiac ischemia, osteoporosis, chronic wounds, diabetes, neural degenerative diseases, neural injuries, bone or cartilage injuries, ablated bone marrow, anemia, liver disease, allopecia, tooth growth, retinal disease or injuries, ear diseases or injury, muscle degeneration, muscle injury, perceived cosmetic deficiencies, organ support, burns and wounds.
25. A method of generating revenue comprising: obtaining a tissue sample from an individual, determining the cell markers that are resident on the cells from the tissue, classifying the stem and progenitor cells, identifying the number of cells in each stem and progenitor cell type, and incorporating the information into a stem and progenitor cell profile, and charging a fee.
26. A method of generating revenue comprising: obtaining a tissue sample from an individual, enriching the sample with stem and progenitor cells, dividing the enriched sample, selectively growing a defined stem cell or progenitor cell line from a divided portion of the enriched sample, counting the grown colonies of the defined stem cell or progenitor cell line and incorporating the information into a stem and progenitor cell profile, and charging a fee.
27. A method of generating revenue comprising: obtaining a tissue sample from an individual and dividing the sample into at least two portions, determining the cell markers that are resident on the cells from the tissue of a first portion of the tissue, classifying the stem and progenitor cells, identifying the number of cells in each stem and progenitor cell type, and incorporating the information obtained from the first portion into a stem and progenitor cell profile; enriching a second portion of the sample with stem and progenitor cells, dividing the enriched sample, selectively growing a defined stem cell or progenitor cell line from a portion of the enriched sample, counting the grown colonies of the defined stem cell or progenitor cell line and incorporating the information into the stem and progenitor cell profile, and charging a fee.
28. The method of generating revenue of any of Claims 25-27, wherein the fee is charged for tendering the profile to the individual.
29. A method of generating revenue comprising: obtaining a stem and progenitor cell profile from an individual, comparing the profile to a database comprising information from a series of stem cell and progenitor cell profiles correlated with the health conditions associated with the profiles, identifying health conditions associated with specific stem and progenitor cell profiles, providing access to the health information, and charging a fee.
30. A database comprising stem and progenitor cell profiles and health conditions in association such that the health conditions are derived from individuals who originated the stem and progenitor cell profiles.
31. A method of generating revenue comprising: obtaining a database comprising stem and progenitor cell profiles in association with health conditions such that the health conditions are derived from individuals who originated the stem and progenitor cell profiles of Claim 11, providing database information to a third party, and charging a fee for the information in the database.
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