WO2011069117A1 - Procédé pour isoler des populations de cellules souches du sang périphérique en procédant à une séparation basée sur leur taille (élutriation) - Google Patents

Procédé pour isoler des populations de cellules souches du sang périphérique en procédant à une séparation basée sur leur taille (élutriation) Download PDF

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Publication number
WO2011069117A1
WO2011069117A1 PCT/US2010/058974 US2010058974W WO2011069117A1 WO 2011069117 A1 WO2011069117 A1 WO 2011069117A1 US 2010058974 W US2010058974 W US 2010058974W WO 2011069117 A1 WO2011069117 A1 WO 2011069117A1
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Prior art keywords
cells
flow rate
peripheral blood
target cell
cell population
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PCT/US2010/058974
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English (en)
Inventor
Wayne Marasco
Satish Medicetty
Yajuan Jiang
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Neostem, Inc.
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Priority to TW099142523A priority Critical patent/TW201130978A/zh
Publication of WO2011069117A1 publication Critical patent/WO2011069117A1/fr

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    • 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
    • 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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood

Definitions

  • the subject of the present invention generally relates to cellular separations employing devices having specificity for cell size and density.
  • the biological sample for the cellular source may be blood, such as mobilized peripheral blood and the like.
  • the present invention relates to a system and method for separating particles, and, in particular, provide advantageous methods for separating peripheral blood into desired subsets of stem cell populations.
  • the adult stem cell is an undifferentiated cell that is found in a differentiated tissue. It has the ability to renew itself and become specialized to yield all the cell types of the tissue from which it originated and, in the appropriate environment, can also become a specialized cell of a different tissue.
  • Adult stem cells are capable of self-renewal for the lifetime of the organism.
  • Sources of adult stem cells have been found in bone marrow, the blood stream, cornea and retina of the eye, the dental pulp of the tooth, liver, skin, gastrointestinal tract, adipose tissue and pancreas.
  • Stem cells offer the possibility of a renewable source of replacement cells and tissues to treat a myriad of diseases, conditions and disabilities.
  • Adult stem cells are relatively quiescent cells, particularly in organisms where cell turnover is low, yet they can mount a rapid and strong response to tissue stress and injury.
  • stem cells As cells designed to withstand crisis and orchestrate repair, stem cells must be especially resilient. Until recently, it had been thought that a stem cell collected from the bone marrow or peripheral blood (a hematopoietic stem cell) could not give rise to cells of a different tissue type, such as nerve cells. However, a number of clinical studies over recent years have affirmed the phenomenon known as plasticity. Plasticity is the ability of adult stem cells to differentiate to other cell types from a specific cell line. In other words, stem cells, from the bone marrow or from the circulating blood do have the ability to differentiate into other cell types such as heart cells or nerve cells.
  • stem cells are unable to find a healthy match and there is a very low chance of finding a suitable donor. Therefore, it would be advantageous to harvest stem cells from these subjects prior to the need for the bone marrow transplantation arising, where the stem cells can be stored for autologous transplantation at a future timepoint. Accordingly, it is useful to harvest stem cells from healthy subjects when the stem cells are unlikely to have been damaged or programmed for disease. Additionally, stem cells could be harvested from any healthy adult human for future autologous transplantation for a disease or as a regenerative therapy, as well as from adults with a positive family history of cardiac disease, diabetes, cancer, neurologic diseases or autoimmune disorders for future autologous therapeutic use.
  • DEP force effectively maps biophysical properties into a translational force whose direction and magnitude reflects cellular properties
  • DEP force may induce separation between particles of different characteristics.
  • DEP has been used on a microscopic scale to separate bacteria from erythrocytes (Markx et al., 1994), viable from nonviable yeast cells (Wang et al, 1993), and erythroleukemia cells from erythrocytes (Huang et al., 1992).
  • the differences in the electrical polarizabilities of the cell types in those various mixtures were greater than those to be expected in many typical cell sorting applications.
  • FFF Field flow fractionation
  • the technique can be used to separate many different types of matter, from a size of about 1 nm to more than about 100 micrometers, which may include, for example, biological and non-biological matter. Separation according to field flow fractionation occurs by differential retention in a stream of liquid flowing through a thin channel.
  • the FFF technique combines elements of chromatography, electrophoresis, and ultracentrifugation, and it utilizes a flow velocity profile established in the thin channel when the fluid is caused to flow through the chamber. Such velocity profile may be, for example, linear or parabolic.
  • a field is then applied at right angles to the flow and serves to drive the matter into different displacements within the flow velocity profile.
  • the matter being displaced at different positions within the velocity profile will be carried with the fluid flow through the chamber at differing velocities.
  • Fields may be based on sedimentation, crossflow, temperature gradient, centrifugal forces, and the like. The technique suffers, however, from producing insufficiently pure cell populations, being too slow, or being too limited in the spectrum of target cells or other matter.
  • stem cells can be enriched from peripheral blood.
  • Whole blood consists of various liquid components and particle components.
  • the liquid portion of blood is largely made up of plasma, and the particle components include red blood cells (erythrocytes) (RBCs), white blood cells (leukocytes) (WBCs), and platelets (thrombocytes) and stem cells, such as but not limited to, hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and other small stem cells such as Very Small Embryonic- like (VSEL) stem cells.
  • HSCs hematopoietic stem cells
  • MSCs mesenchymal stem cells
  • VSEL Very Small Embryonic- like stem cells.
  • these constituents have similar densities, their average density relationship, in order of decreasing density, is as follows: red blood cells, white blood cells, platelets, and plasma.
  • the particle components are related according to size, in order of decreasing size, as follows: white blood cells, red blood cells, and platelets
  • Peripheral blood and mobilized peripheral blood comprise many different types of cells, including stem cells such as, but not limited to, Very Small Embryonic Like Stem cells (VSELs), mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs).
  • VSELs Very Small Embryonic Like Stem cells
  • MSCs mesenchymal stem cells
  • HSCs hematopoietic stem cells
  • Conventional methods to isolate stem cell populations from the peripheral blood use positive and negative selection methods, e.g., using cell-surface markers in methods such as fluorescence activated cell sorting (FACS) and other immunoselection methods to select or exclude a cell population. Additionally, such methods also typically isolate only one stem cell population at a time from the peripheral blood sample.
  • FACS fluorescence activated cell sorting
  • the present invention relates to a method for isolating multiple populations of stem cells in at least one step from a single peripheral blood or mobilized peripheral blood sample without using positive or negative selection, therefore enabling a "no-touch" method to isolate different stem cell populations without contaminating the stem cell populations with positive selection agents (e.g., antibodies, etc).
  • the present invention relates to a method for the flow-rate separation of stem cell populations using elutriation (sized-based separation) to negatively exclude cells based on size, to separate the peripheral blood into various elutriation fractions, each comprising a different stem cell of interest.
  • the inventors have demonstrated that different fractions or subtractions comprise an increase in yield of Very Small Embryonic Like Stem cells (VSELs), MSCs or HSCs as compared to non-fractionated apheresis product.
  • VSELs Very Small Embryonic Like Stem cells
  • MSCs Magnetic Cell Sorting
  • HSCs High Capacity Cell Sorting
  • the isolated stem cell populations obtained by the methods as disclosed herein provide a library of different stem cell types from a particular individual which can be maintained in culture and/or cryopreserved for future use, e.g., for use alone, or selectively recombined (e.g., custom mixing) for individualized autologous therapeutic applications in regenerative therapy.
  • the isolated stem cell populations also provide a pool of different stem cell populations for personalized cell-based assays to assess the effect of a persons diet, pharmacogenetics, neutrochemicals, and lifestyle on the function and viability of different stem cell populations, either alone or as a combination of different stem cells.
  • the present invention generally relates to methods and compositions for the separation or fractionation of peripheral blood into a plurality of fractions which comprise different stem cell populations from a single peripheral blood sample from a subject in a single collection process.
  • the present invention generally relates to a quick, easy, effective and inexpensive method for isolating stem cell populations from peripheral blood, such as peripheral blood obtained from a human subject without using positive selection techniques.
  • peripheral blood such as peripheral blood obtained from a human subject without using positive selection techniques.
  • the peripheral blood is mobilized peripheral blood, and in some embodiments, the peripheral blood is pre-processed by aphaeresis to generate an apheresis product.
  • the present invention relates to methods for isolating a plurality of different stem cell populations from a peripheral blood sample without the need to add positive selection agents to the peripheral blood sample.
  • the inventors have discovered a method to isolate a plurality of different stem cell populations from a single sample without contaminating the collected stem cell populations with positive selection agents.
  • the present invention provides methods to fractionate peripheral blood into a plurality of fractions, wherein each fraction comprises a distinct target population of stem cells.
  • the present invention relates to a method for enriching for a target population of stem cells obtained from peripheral blood without using positive selection techniques.
  • the present invention relates to a method of separating a peripheral blood sample into a plurality of different fractions, enabling the enrichment of a plurality of different target stem cell populations in different fractions.
  • the resulting fractions comprising a target stem cell population can be further separated into subsets or subpopulations of desired target stem cells, if desired.
  • the collected fraction comprising the target stem population can be subject to positive selection methods to enrich for the desired stem cell population.
  • BM bone marrow
  • PB peripheral blood
  • Peripheral blood comprises a variety of different types of stem cells, including hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and other stem cell populations, including but not limited to Very Small Embryonic Like (VSEL) stem cells.
  • HSCs hematopoietic stem cells
  • MSCs mesenchymal stem cells
  • VSEL Very Small Embryonic Like stem cells.
  • Typical conventional methods to isolate such stem cell populations, such as HSCs, MSCs and VSELs, from peripheral blood samples involve positive selection methods.
  • selection methods include methods based on cell-surface markers, e.g., immunoselection, which involves separation and isolation based on the binding of an affinity molecule such as an antibody or cell-surface ligand to the surface of the target stem cell to be separated, where the antibody is used to isolate the target stem cell.
  • an affinity molecule such as an antibody or cell-surface ligand
  • Such positive separation methods are commonly known in the art, and include, without limitation, fluorescence cell sorting (FACS), immuodensity separation, immunomagnetic separation and the like.
  • FACS fluorescence cell sorting
  • immuodensity separation immuodensity separation
  • immunomagnetic separation immunomagnetic separation and the like.
  • selection agents such as antibodies
  • selection methods are also expensive, time consuming and slow procedures that often require skilled professionals or specific training of technicians and/or specialized equipment.
  • conventional selection methods to isolate stem cells populations from peripheral blood samples often only enrich for one target stem cell population from a peripheral blood sample and discard other potentially useful non-selected, or non-enriched stem cell populations which are also present in the peripheral blood sample.
  • the present invention directed to a method to isolate or enrich for numerous stem cell populations from a peripheral blood sample without using positive selection techniques as disclosed herein, has numerous advantages over existing methods for stem cell enrichment from peripheral blood, including, but not limited to, (i) enriching for multiple different stem populations from a single peripheral blood sample, enabling the collection of a variety of different stem cell populations present in peripheral blood and avoiding having to discard potentially valuable or useful stem cell populations, (ii) enriching for stem cell populations in the peripheral blood without the need to add additional agents, therefore avoiding contamination of collected stem cell population or the peripheral blood sample with agents, (e.g., agents which may not meet with FDA approval should the enriched stem cell population be subsequently used in autologous or allogenic transplantation in humans), (iii) providing a method to enrich for multiple different stem cell populations from peripheral blood which can be readily carried out by personnel of ordinary skill with little or no training, which uses a quick, easy and inexpensive method, and can be readily carried out in multiple locations without the need for specialized equipment, reagent
  • the present invention provides methods for the isolation of a plurality of different stem cell populations without the contamination of agents typically used for positive selection or isolation of stem cell populations, e.g., without contamination with antibodies or other agents used in immunoselection methods, or without the need to isolate and select the cells based on cell-surface markers, such as done in conventional stem cell isolation methods that use immunoselection, such as fluorescent cell sorting (FACS) or magnetic bead separation after interaction of the target cell with a labeled antibody.
  • FACS fluorescent cell sorting
  • magnetic bead separation after interaction of the target cell with a labeled antibody such as fluorescent cell sorting (FACS) or magnetic bead separation after interaction of the target cell with a labeled antibody.
  • Immunoselection methods include affinity methods with antibodies labeled to magnetic beads, biodegradable beads, non-biodegradable beads and antibodies panned to surfaces including dishes and combinations of such methods.
  • the present invention generally relates to peripheral blood fractionation comprising the separation of cells present in a peripheral blood (PB) sample into distinct fractions of cells, where each fraction is heterogenous population of cells, where some fractions comprise at least one distinct target stem cell population.
  • each PB fraction comprising a heterogenous population of cells can be further fractionated, for example into distinct homogenous target stem cell populations.
  • a PB fraction comprising a heterogenous population of cells which comprises at least two populations of target stem cell populations e.g. HSCs and MSCs
  • HSCs and MSCs can be further fractionated onto a substantially pure population of HSCs, a substantially pure population of MSCs and a heterogeneous population of cells which are neither HSCs or MSCs.
  • a PB fraction comprising a heterogonous population of cells which comprises a population of target stem cells (e.g., VSEL stem cells) can be further fractionated onto a substantially pure population of VSELs and a heterogeneous population of non-VSEL cells.
  • VSEL stem cells e.g., VSEL stem cells
  • the present invention relates to methods for the isolation of a plurality of target stem cell populations from a single peripheral blood sample without using positive selection techniques, and thus avoiding contaminating the isolated target stem cell population or the peripheral blood sample with an agent which would otherwise be necessary for the selection or enrichment of a target stem cell population.
  • the peripheral blood fractionation is achieved by elutriation, wherein the flow rate through the elutriation apparatus determines which cells from the peripheral blood are collected in a specific PB fraction.
  • an apheresis product from peripheral blood is processed at different flow rates through an ELUTRA® apparatus, or other suitable elutriation apparatus or machine.
  • the flow rates used in the elutriation procedure is maintained and centripital acceleration force (i.e., "g") is varied to fractionate the peripheral or apheresed blood.
  • a PB fraction collected from a flow rate of 50ml/minute comprises a target stem cell population comprising VSEL stem cells, as disclosed in the Examples herein.
  • the percentage of VSELs in a PB fraction obtained from a flow rate of 50ml/minute is greater than about 0.1%, or about 0.2%, or about 0.3% or about 0.4% or about 0.5%, or greater than 0.5%.
  • the PB fraction collected from a flow rate of 50ml/minute is enriched in VSELs by about 15-fold, or about 20-fold, or about 30-fold, or about 40-fold, or about 50- fold or greater than about 50-fold as compared to non-fractionated peripheral blood.
  • a PB fraction collected from a flow rate of greater or equal to about 90ml/min comprises a target stem cell population comprising HSCs and MSCs, as disclosed in the Examples herein.
  • the percentage of HSCs or MSCs in a PB fraction obtained from a flow rate of greater or equal to about 90ml/min is greater than about 15%, or about 20%, or about 30% or about 40% or about 50% or greater than about 50%, or any integer between 15% and 50%, or above about 50%.
  • the present invention provides a method for separating peripheral blood to obtain at least four target cell populations without using positive selection techniques, comprising: (a) flowing a peripheral blood sample from a subject through a fluid chamber of an elutriation apparatus, wherein a first flow rate is selected wherein cells in the peripheral blood larger than the smallest cells will flow through, and smaller cells are retained in the fluid chamber; (b) collecting the retained small cells from step (a) in the fluid chamber without subjecting the retained cells to a positive selection step, to obtain a first target cell population; (c) recirculating the peripheral blood sample comprising the non-retained cells through the fluid chamber of the elutriation apparatus, and increasing the flow rate of the remaining peripheral blood sample, wherein cells in the peripheral blood larger than the smallest cells will flow through, and smaller cells are retained in the fluid chamber; (d) collecting the retained small cells in the fluid chamber from step (c) without subjecting the retained cells to a positive selection step, to obtain a second target cell population; and (e) repeat
  • the first flow rate is 20ml/minute or less and the first target cell population comprises platelets.
  • an increased flow rate is 50ml/minute or less and the target cell population comprises a population of Very Small Embryonic-like (VSEL) stem cells.
  • an increased flow rate is 70ml/minute or less and the target cell population comprises a population of red blood cells (RBC).
  • an increased flow rate is 90ml/minute or less and the target cell population comprises a population of white blood cells (WBC).
  • an increased flow rate is 105ml/minute or less, and the target cell population comprises a population of hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs).
  • HSCs hematopoietic stem cells
  • MSCs mesenchymal stem cells
  • the methods as disclosed herein do not use a positive selection technique such as immunoselection or immunodensity selection, e.g., FACS. In some embodiments, the methods as disclosed herein do not use a positive selection technique which uses antibodies, or antibody fragments.
  • a retained cell population e.g., a fraction of PB which comprises a target stem cell population
  • positive selection techniques to enrich for a desired target population of stem cells in the retained cell population.
  • the method for separating peripheral blood to obtain at least four target cell populations as disclosed herein uses a peripheral blood sample, such as a human peripheral blood sample, including a mobilized peripheral blood sample.
  • a peripheral blood sample is obtained from a human subject after administration of a mobilizing agent, such as, for example, where a human subject has been administered a mobilizing agent for at least 2 days, or four days or less.
  • the peripheral blood sample is an apheresis product.
  • One aspect of the present invention relates to a method for separating peripheral blood to obtain at least four target cell populations without using positive selection techniques comprising: (a) flowing a peripheral blood sample from a subject through a fluid chamber of an elutriation apparatus, wherein a first flow rate is selected wherein cells in the peripheral blood larger than the smallest cells will flow through, and smaller cells are retained in the fluid chamber; (b) collecting the retained small cells from step (a) in the fluid chamber without subjecting the retained cells to a positive selection step, to obtain a first target cell population; (c) recirculating the peripheral blood sample comprising the non-retained cells through the fluid chamber of the elutriation apparatus, and increasing the flow rate of the remaining peripheral blood sample, wherein cells in the peripheral blood larger than the smallest cells will flow through, and smaller cells are retained in the fluid chamber; (d) collecting the retained small cells in the fluid chamber from step (c) without subjecting the retained cells to a positive selection step, to obtain a second target cell population; and (e) repeat
  • the first flow rate is 20ml/minute or less and the first target cell population comprises platelets.
  • an increased flow rate is 50ml/minute or less and the target cell population comprises a population of Very Small Embryonic-like (VSEL) stem cells.
  • an increased flow rate is 70ml/minute or less.
  • an increased flow rate is 90ml/minute or less and the target cell population comprises a population of red blood cells (RBC) and white blood cells (WBC).
  • RBC red blood cells
  • WBC white blood cells
  • an increased flow rate is greater than 90ml/minute or less, and the target cell population comprises a population of hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs).
  • an increased flow rate is about 105ml/minute or less, and the target cell population comprises a population of hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs).
  • the methods as disclosed herein do not use a positive selection technique, such as immunoselection or immunodensity selection, e.g., FACS. In some embodiments, the methods as disclosed herein do not use a positive selection technique that uses antibodies or antibody fragments.
  • a positive selection technique such as immunoselection or immunodensity selection, e.g., FACS. In some embodiments, the methods as disclosed herein do not use a positive selection technique that uses antibodies or antibody fragments.
  • a retained cell population can be subjected to negative selection techniques to enrich for a desired target population of stem cells in the retained cell population.
  • the negative selection techniques use immunoselection and immunodensity selection methods.
  • the negative selection techniques are used to remove undesired cells and components from the target stem cell population.
  • the method can be used to obtain a plurality of stem cell populations from a peripheral blood sample such as a human peripheral blood sample, including a mobilized peripheral blood sample.
  • a peripheral blood sample is obtained from a human subject after administration of a mobilizing agent, for example, where a human subject has been administered a mobilizing agent for at least 1 day, but less than four days.
  • a mobilizing agent is administered for two days.
  • the mobilizing agent is administered for three days.
  • the peripheral blood sample is an apheresis product.
  • Another aspect of the present invention relates to a method for treating a disease or disorder in a subject with at least one autologous stem cell population comprising: (a) utilizing at least one stem cell population from a peripheral blood sample, wherein the peripheral blood sample is obtained from a subject who has been administered a mobilizing agent for 4 days or less, and wherein the at least one stem cell population was enriched using a flow-rate separation process without using a positive selection technique; and (b) administering at least one stem cell population to the subject to treat the disease or disorder.
  • At least one stem cell population obtained using the methods as disclosed herein has been cryopreserved.
  • at least one stem cell population comprises non-stem cells, for example, a stem cell population comprising at least 10% of a target stem cell population.
  • at least one stem cell population is a substantially pure population of a target stem cell population.
  • at least one stem cell population has been expanded in vitro prior to administering to the subject, such as a human subject.
  • the methods enable collecting multiple stem cell populations from peripheral blood, wherein at least one stem cell population is Very Small Embryonic-like (VSEL) stem cells, and at least one stem cell population is MSCs, and at least one stem cell population is HSCs.
  • at least one stem cell population administered to the subject is a combination of cells comprising any combination selected from the group of Very Small Embryonic-like (VSEL) stem cells, MSCs or HSCs.
  • the present invention provides a method for separating a target cell population from peripheral blood comprising:
  • steps (a), (b), and (c) do not comprise the use of positive selection techniques.
  • the first flow rate is selected so that cells in the peripheral blood sample smaller than Very Small Embryonic Like stem cells (VSELs) flow through the fluid chamber and VSELs are retained in the fluid chamber and (ii) the second flow rate is selected so that VSELs flow through the fluid chamber.
  • VSELs Very Small Embryonic Like stem cells
  • the first flow rate is about 35 ml/min
  • the second flow rate is about 50-70 ml/min
  • VSELs are collected.
  • the first flow rate is selected so that cells in the peripheral blood sample smaller than mesenchymal stem cells (MSCs) flow through the fluid chamber and
  • MSCs are retained in the fluid chamber and (ii) the second flow rate is selected so that MSCs flow through the fluid chamber.
  • the first flow rate is about 100 ml/min
  • the second flow rate is about 110-120 ml/min
  • MSCs are collected.
  • the first flow rate is selected so that cells in the peripheral blood sample smaller than hematopoietic stem cells (HSCs) flow through the fluid chamber and
  • HSCs are retained in the fluid chamber and (ii) the second flow rate is selected so that HSCs flow through the fluid chamber.
  • the first flow rate is about 90 ml/min
  • the second flow rate is about 100 ml/min
  • HSCs are collected.
  • the positive selection technique is immunoselection or
  • the peripheral blood sample is a human peripheral blood sample.
  • the peripheral blood sample is a mobilized peripheral blood sample.
  • the peripheral blood sample is obtained from a subject who has been administered a mobilizing agent for 4 days or less.
  • the collected target cell population is cryopreserved.
  • At least one of the collected target cell populations is administered to a subject.
  • At least one of the collected target cell populations are administered to a subject from whom the peripheral blood was originally obtained.
  • At least one of the collected target cell populations has been expanded in vitro prior to administering to the subject.
  • the subject is a human subject.
  • At least one of the collected target cell populations is VSELs.
  • At least one of the collected target cell populations is MSCs.
  • At least one of the collected target cell populations is HSCs.
  • the present invention provides a method for separating target cell populations from peripheral blood comprising:
  • steps (a) and (b) do not comprise the use of positive selection techniques.
  • the method further comprises:
  • step (c) does not comprise the use of positive selection techniques.
  • the method further comprises:
  • step (d) does not comprise the use of positive selection techniques.
  • the method further comprises:
  • step (e) does not comprise the use of positive selection techniques.
  • the method further comprises:
  • step (f) does not comprise the use of positive selection techniques.
  • the method further comprises:
  • step (g) does not comprise the use of positive selection techniques.
  • the first flow rate is selected so that the first target cell population is cells smaller than Very Small Embryonic Like stem cells (VSELs) and (ii) the second flow rate is selected so that the second target cell population is VSELs.
  • VSELs Very Small Embryonic Like stem cells
  • the first flow rate is about 35 ml/min
  • the second flow rate is about 50-70 ml/min
  • VSELs are collected.
  • the third flow rate is selected so that the first target cell population is cells smaller than hematopoietic stem cells (HSCs) and (ii) the fourth flow rate is selected so that the second target cell population is HSCs.
  • HSCs hematopoietic stem cells
  • the third flow rate is about 90 ml/min
  • the fourth flow rate is about 100 ml/min
  • HSCs are collected.
  • the fourth flow rate is selected so that the first target cell population is cells smaller than mesenchymal stem cells (MSCs) and (ii) the fifth flow rate is selected so that the second target cell population is MSCs.
  • the third flow rate is about 100 ml/min
  • the fourth flow rate is about 110-120 ml/min
  • MSCs are collected.
  • the first flow rate is about 35 ml/min and the first target cell population is platelets;
  • the second flow rate is about 50 ml/min and the second target cell population is VSELs;
  • the third flow rate is about 70 ml/min and the third target cell population is VSELs;
  • the fourth flow rate is about 90 ml/min and the fourth target cell population is red blood cells;
  • the fifth flow rate is about 100 ml min and the fifth target cell population is HSCs;
  • the sixth flow rate is about 110 ml min and the sixth target cell population is MSCs;
  • the seventh flow rate is about 120 ml/min and the seventh target cell population is MSCs.
  • the positive selection technique is immunoselection or
  • the peripheral blood sample is a human peripheral blood sample.
  • the peripheral blood sample is a mobilized peripheral blood sample.
  • the peripheral blood sample is obtained from a subject who has been administered a mobilizing agent for 4 days or less.
  • the collected target cell population is cryopreserved.
  • At least one of the collected target cell populations is administered to a subject.
  • At least one of the collected target cell populations are administered to a subject from whom the peripheral blood was originally obtained. [0080] In some embodiments, at least one of the collected target cell populations has been expanded in vitro prior to administering to the subject.
  • the subject is a human subject.
  • At least one of the collected target cell populations is VSELs.
  • At least one of the collected target cell populations is MSCs.
  • At least one of the collected target cell populations is HSCs.
  • the present invention provides a method for separating target cell populations from peripheral blood comprising:
  • step (c) optionally, repeating step (b) until a desired number of target cell populations are collected;
  • steps (a), (b), and (c) do not comprise the use of positive selection techniques.
  • the present invention provides VSELs, MSCs, or HSCs product by one of the methods disclosed above.
  • FIG. 1 shows a graph displaying that Very Small Embryonic-like Stem Cells (VSELs) can be enriched by size-based separation (elutriation) of various cell fractions in the apheresis product.
  • VSELs Very Small Embryonic-like Stem Cells
  • Fraction 1 Flow rate 20 mL/min
  • Fraction 2 50 mL/min
  • Fraction 3 70 mL/min
  • Fraction 4 90 mL/min
  • Fraction 5 Fraction 5 (>90 mL/min).
  • Fraction 2 is highly enriched in VSELs and can be used to harvest purified populations of VSELs for clinical applications.
  • Figure 2 is a photograph showing the size-based separation of an apheresis product from peripheral blood into 5 separate fractions.
  • Figure 3A-F shows the enrichment of MSCs from apheresed human peripheral blood.
  • G- CSF mobilized apheresed blood was separated using an ELUTRA® Cell Separation System. MSCs were enriched in fractions 4 and 5.
  • Figure 3F Fraction 6.
  • Figure 4A-E shows cells from elutriation fractions that were plated in a CFU-F assay. Colonies of adherent cells developed from fractions 4 and 5.
  • Figure 5A-B shows the mobilization of MSCs into peripheral blood. Patients received two consecutive shots of G-CSF subcutaneously. The degree of mobilization of mesenchymal stem cells was monitored at day 0 prior to receiving a G-CSF injection, day 3 after receiving the second G-CSF injections, and in the apheresis product.
  • Figure 5A MSC mobilization assessed by evaluation of CD457CD317CD1057CD73 + cells.
  • Figure 6A-B shows FACS analysis of MSC phenotype in ELUTRA® fractions.
  • Figure 6A Fractions 4 and 5 were enriched for CD457CD105" cells compared to other fractions.
  • Figure 6B Fractions 4 and 5 showed an increase in the proportion of CD457CD105 + , CD45 " /CD90 " , and CD271 + cells as compared to the aphesis product starting population.
  • the present invention enables flexibility to optimize the recovery of specific target stem cell populations from a peripheral blood sample, where a plurality of different target stem cell populations are enriched with yields that have a high number of cells with low cross-cellular contamination of non-stem cells or non-target stem cells.
  • One aspect of the present invention relates to the use of elutriation to rapidly and reliably separate bulk stem cell populations from peripheral blood, where each target stem cell population has a different sedimentation coefficient (size and density) as to other distinct populations of target stem cells, allowing enrichment of different target stem cell populations with virtually no loss in either recovery or viability of the target stem cell populations.
  • target stem cells obtained from peripheral blood according to the methods as disclosed herein are useful for cell therapy.
  • stem cell therapies there is a need to use bone marrow (BM)- or peripheral blood (PB)-derived stem cells for cell replacement or regenerative therapies.
  • BM bone marrow
  • PB peripheral blood
  • Methods for separating stem cells and non-stem cells can be divided into three basic types:
  • (a) separation based on a cell's physical properties such as cell density and size which includes methods based on differential migration in a fluid flow (field flow fractionation) or in a sedimentation field (centrifugation) or in a fluid flow in the presence of a sedimentation field (elutriation).
  • Differential sedimentation can be performed in combination with a solution or hydrogels of varying inherent densities, either in discontinuous or continuous (gradient) form.
  • separation based on specific surface molecular groups include use of affinity monoclonal or polyclonal antibodies to cell surface receptors or other determinants. Affinity methods involve negative selection (e.g., interaction with the cell surface receptor and the affinity monoclonal antibody to remove unwanted populations of cells) or positive selection (interaction with the cell surface receptors and the affinity molecule, e.g., monoclonal antibody to select for the desired target cell).
  • properties e.g., selection based on density gradient media used to isolate a population of cells in combination with cell-surface specific antibodies to recognize target cells or an undesired subpopulation of the cells.
  • isolation of stem cells has typically been based on cell-surface properties.
  • conventional methods to isolate stem cell populations, such as HSCs, MSCs and VSELs, from a peripheral blood sample typically involve cell-surface based separation methods, such as immunoselection, where separation and isolation of a particular stem cell population is based on the binding of an affinity molecule, such as an antibody or cell-surface ligand to the surface of the target stem cell to be separated .
  • separation methods are commonly known in the art, and include without limitation, fluorescence cell sorting (FACS), mnuuodensity separation and immunomagnetic separation and the like.
  • FACS fluorescence cell sorting
  • mnuuodensity separation mnuuodensity separation
  • immunomagnetic separation and the like.
  • the invention provides a method for separating desired populations of stem cells that can be performed using large amounts of input cells without the need for a selection process using cell surface markers or reagents associated with such a process.
  • the instant invention provides separation parameters and demonstrates that several types of stem cells can be effectively enriched from other cellular components of peripheral and apheresed blood.
  • the resulting fractions comprising a target stem cell population can be further separated into subsets of desired cells for collection, if desired.
  • Kwekkeboom et al. do not discuss the use of CCE for separation of different stem cell populations from a peripheral blood sample. More particularly, Kwekkeboom found no indication of preferential co-elutriation of primitive stem cells with any fraction, including small cell fractions.
  • the term "apheresis” as used herein refers to the process or procedure in which blood is drawn from a donor subject and separated into its components, some of which are retained, such as plasma, platelets and/or stem cell populations, and the remainder returned by transfusion to the donor subject. This process can also be referred to in the art as
  • apheresis The forms of apheresis include: Plasmapheresis ⁇ to harvest plasma (the liquid part of the blood); Leukapheresis— to harvest leukocytes (white blood cells); Granulocytapheresis— to harvest granulocytes (neutrophils, eosinophils, and basophils); Lymphocytapheresis— to harvest lymphocytes; Lymphoplasmapheresis - to harvest lymphocytes and plasma; Plateletpheresis (thrombocytapheresis) - to harvest platelets (thrombocytes). Apheresis takes longer than a whole blood donation. A whole blood donation takes about 10-20 minutes to collect the blood, while an apheresis donation may take about 1- 2 hours.
  • apheresis product refers to the heterogeneous population of cells collected from the process of apheresis. As disclosed herein, the cells present in an apheresis product may be separated using elutriation.
  • exclusion refers to the separation or enrichment of cells, e.g., stem cells from peripheral blood on the basis of their differential sedimentation rate.
  • Elutriation as used herein is a noninvasive method for separating large numbers of cells on the basis of their size and mass. Also, elutriation allows for separating mixed populations of cells, in particular stem cells at different stages of the cell division cycle without perturbing stem cell metabolism or using synchronizing agents
  • enriching or “enriched” or “enrich” are used interchangeably herein and mean that the yield (i.e., fraction) of cells of one type is increased by at least 10% over the fraction of cells of that type in the starting culture or preparation.
  • selection refers to isolating different cell types into one or more populations and collecting the isolated population as a target cell population which is enriched in a specific target stem cell population. Selection can occur using positive selection for the enriched cell population or negative selection to discard non-target cell populations.
  • positive selection refers to a method where the desired stem cells are targeted for selection, e.g., using a monoclonal antibody to a specific cell surface antigen on the desired or target stem cell.
  • negative selection refers to targeting unwanted or non-target stem cells for depletion, e.g., using monoclonal antibodies to specific cell surface antigens. In negative selection, desired cells or target cells are not labeled with antibody.
  • the term "remove” as used herein refers to separate and select and set aside (e.g., for retaining or discarding). Thus, a target population of cells can be removed from a mixed population of cells with the intent of keeping them or discarding them.
  • immunosensing refers to a process where cells, e.g., stem cells, are labeled using monoclonal antibodies and separated by selecting for the antibody, using a marker on the antibody, such as fluorescence, or magnetic particles.
  • immunodensity selection refers to a process where unwanted cells, e.g., non-target cells are targeted with monoclonal antibody-based reagents, and pellet when centrifuged over density medium.
  • fractionation refers to the production of homogeneous sets of stem cell populations from a population of heterogeneous stem cells.
  • peripheral blood refers to whole blood obtained from a subject.
  • the terms "mobilized peripheral blood” as used herein refers to peripheral blood which is obtained from a subject where the subject has been administered a mobilizing agent for enhancing the number of stem cells in the peripheral blood, by increasing the migration of stem cells from the bone marrow (e.g., increasing BM-derived stem cells) into the peripheral blood, or increasing the proliferation of stem cell present in the peripheral blood (e.g..
  • Mobilization may be effectuated in a subject by administering an effective amount of a mobilizing agent, e.g., by a combination of - 11 chemoattractants (e.g., cytokines) and loss of adhesiveness of pools or populations of stem cells residing in stem cell niches in peripheral tissues and the bone marrow.
  • a mobilizing agent e.g., by a combination of - 11 chemoattractants (e.g., cytokines) and loss of adhesiveness of pools or populations of stem cells residing in stem cell niches in peripheral tissues and the bone marrow.
  • An "effective amount” is an amount of a mobilizing agent sufficient to effect a significant increase in the number and/or frequency of stem cells in the peripheral blood.
  • An effective amount can be administered in one or more administrations, applications or dosages.
  • a therapeutically effective amount of a mobilizing agent depends on the mobilizing agent selected, e.g., G-CSF or GM-CSF.
  • the mobilizing agent such as G-CSF or GM-CSF
  • the mobilizing agent can be administered from one or more times per day to one or more times per week; including once every other day.
  • the skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to, previous treatments, the general health and/or age of the subject, and whether other diseases are present.
  • treatment of a subject with a therapeutically effective amount of the G-CSF or GM-CSF can include a single treatment or a series of treatments.
  • Mobilization refers to the process whereby the cells leave the bone marrow and enter the blood. Mobilization may be effectuated by a combination of chemoattractants (e.g., cytokines) and loss of adhesiveness of pools or populations of stem cells residing in stem cell niches in peripheral tissues and the bone marrow.
  • chemoattractants e.g., cytokines
  • stem cells is used in a broad sense and includes traditional stem cells, progenitor cells, preprogenitor cells, reserve cells, and the like.
  • stem cell or “progenitor” are used interchangeably herein, and refer to an undifferentiated cell which is capable of proliferation and of giving rise to more progenitor cells having the ability to generate a large number of mother cells that can in turn give rise to differentiated, or differentiable, daughter cells.
  • the daughter cells themselves can be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential.
  • stem cell refers then, to a cell w ith the capacity or potential, under particular circumstances, to differentiate to a more specialized or differentiated phenotype, and which retains the capacity, under certain circumstances, to proliferate without substantially differentiating.
  • progenitor or stem cell refers to a generalized mother cell whose descendants (progeny) specialize, often in different directions, by differentiation, e.g., by acquiring completely individual characters, as occurs in progressive diversification of embryonic cells and tissues.
  • Cellular differentiation is a complex process typically occurring through many cell divisions.
  • a differentiated cell may derive from a multipotent cell which itself is derived from a multipotent cell, and so on.
  • stem cells While each of these multipotent cells may be considered stem cells, the range of cell types each can give rise to may vary considerably. Some differentiated cells also have the capacity to give rise to cells of greater developmental potential. Such capacity may be natural or may be induced artificially upon treatment with various factors. In many biological instances, stem cells are also "multipotent” because they can produce progeny of more than one distinct cell type, but this is not required for "sternness.” Self-renewal is the other classical part of the stem cell definition, and it is essential as used in this document. In theory, self-renewal can occur by either of two major mechanisms. Stem cells may divide asymmetrically, with one daughter retaining the stem state and the other daughter expressing some distinct other specific function and phenotype.
  • stem cells in a population can divide symmetrically into two stems, thus maintaining some stem cells in the population as a whole, while other cells in the population give rise to differentiated progeny only.
  • stem cells that begin as stem cells might proceed toward a differentiated phenotype, but then "reverse” and re-express the stem cell phenotype, a term often referred to as "dedifferentiation.”
  • Exemplary stem cells include embryonic stem cells, adult stem cells, pluripotent stem cells, neural stem cells, liver stem cells, muscle stem cells, muscle precursor stem cells, endothelial progenitor cells, bone marrow stem cells, chondrogenic stem cells, lymphoid stem cells, mesenchymal stem cells, hematopoietic stem cells, central nervous system stem cells, peripheral nervous system stem cells, and the like.
  • Descriptions of stem cells, including method for isolating and culturing them, may be found in, among other places, Embryonic Stem Cells, Methods and Protocols, Turksen, ed., Humana Press, 2002; Weisman et al., Annu. Rev. Cell. Dev. Biol. 17:387 403; Pittmger et al. Science, 284:143 47, 1999; Animal Cell Culture, Masters, ed., Oxford University Press, 2000; Jackson et al., PNAS
  • progenitor cell is used herein to refer to a stem cell that has a cellular
  • progenitor cells can give rise to multiple distinct differentiated cell types or to a single differentiated cell type, depending on the developmental pathway and on the environment in which the cells develop and differentiate.
  • totipotent refers to a stem cell that can give rise to any tissue or cell type in the body.
  • Pluripotent stem cells can give rise to any type of cell in the body except germ line cells.
  • stemm cells that can give rise to a smaller or limited number of different cell types are generally termed “multipotent.”
  • multipotent Thus, totipotent cells differentiate into pluripotent cells that can give rise to most, but not all, of the tissues necessary for fetal development.
  • Pluripotent cells undergo further differentiation into multipotent cells that are committed to give rise to cells that have a particular function.
  • multipotent hematopoietic stem cells give rise to the red blood cells, white blood cells and platelets in the blood.
  • pluripotent refers to a cell with the capacity, under different conditions, to differentiate into cell types characteristic of all three germ cell layers
  • Pluripotent cells are characterized primarily by their ability to differentiate to all three germ layers, using, for example, a nude mouse teratoma formation assay. Pluripotency is also evidenced by the expression of embryonic stem (ES) cell markers, although the preferred test for pluripotency is the demonstration of the capacity to differentiate into cells of each of the three germ layers.
  • ES embryonic stem
  • a pluripotent cell is an undifferentiated cell.
  • pluripotency or a “pluripotent state” as used herein refer to a cell with the ability to differentiate into all three embryonic germ layers: endoderm (gut tissue), mesoderm (including blood, muscle, and vessels), and ectoderm (such as skin and nerve), and that typically has the potential to divide in vitro for a long period of time, e.g., greater than one year or more than 30 passages.
  • multipotent when used in reference to a “multipotent cell” refers to a cell that is able to differentiate into some but not all of the cells derived from all three germ layers. Thus, a multipotent cell is a partially differentiated cell. Multipotent cells are well known in the art, and examples of multipotent cells include adult stem cells, such as for example, hematopoietic stem cells and neural stem cells. Multipotent means a stem cell may form many types of cells in a given lineage, but not cells of other lineages. For example, a multipotent blood stem cell can form the many different types of blood cells (red, white, platelets, etc.), but it cannot form neurons. [00127] The term “multipotency” refers to a cell with the degree of developmental versatility that is less than totipotent and pluripotent.
  • totipotency refers to a cell with the degree of differentiation describing a capacity to make all of the cells in the adult body as well as the extra-embryonic tissues including the placenta.
  • the fertilized egg zygote is totipotent as are the early cleaved cells
  • MSC meenchymal stem cell
  • pluripotent stem cells capable of differentiating into more than one specific type of mesenchymal or connective tissue (i.e., tissues of the body which support specialized elements; e.g., adipose, osseous, stroma, cartilaginous, elastic and fibrous connective tissues).
  • mesenchymal stem cells i.e., tissues of the body which support specialized elements; e.g., adipose, osseous, stroma, cartilaginous, elastic and fibrous connective tissues.
  • Human mesenchymal stem cells hMSCs
  • SH2, SH3 and SH4 monoclonal antibodies
  • human MSCs can be identified based on (i) phenotypic marker expression of CD34-, CD45-, CD90+, CD 105+ and CD44+, (ii) functional phenotype, including the ability to form colony forming units in a CFA assay as disclosed in the Examples herein, and ability to differentiate into tissues which support specialized elements, including but not limited to: chondrocytes, cartilage and adipocytes.
  • Other markers expressed by MSCs are known in the art and include without limitation CD71, CD73, Stro-1, and CD166, and CD271. In certain embodiments, MSCs are lin " .
  • VSEL stem cell The term "very small embryonic -like stem cell” is also referred to herein as "VSEL stem cell” and refers to pluripotent stem cells.
  • VSEL stem cells are human VSELs and may be characterized as lin “ , CD45 " , and CD34 + .
  • the VSELs are human VSELs and may be characterized as lin " , CD45 " , and CD133 + .
  • the VSELs are human VSELs and may be characterized as lin " , CD45 " , and CXCR4 + .
  • the VSELs are human VSELs and may be characterized as lin " , CD45 “ , CXC 4 + , CD133 + , and CD34 + .
  • Human VSELs express at least one of SSEA-4, Oct-4, Rex-1, and Nanog, and possess large nuclei surrounded by a narrow rim of cytoplasm, and contain embryonic-type unorganized chromatin. VSELs also have high telomerase activity.
  • the ⁇ 3 ⁇ 4ELs are human VSELs and may be characterized as lin " , CD45 “ , CXCR4 + , CD133 + , Oct 4 + , SSEA4 + , and CD34 + .
  • the human VSELs may be less primitive and may be characterized as lin " , CD45 “ , CXCR4 ⁇ , CD 133 " , and CD34 + .
  • the human VSELs may be enriched for pluripotent embryonic transcription factors, e.g., Oct-4, Sox2, and anog.
  • the human VSELs may have a diameter of 4-5 ⁇ , 4-6 ⁇ , 4-7 urn, 5-6 ⁇ , 5-8 ⁇ , 6-9 ⁇ , or 7-10 ⁇ .
  • peripheral blood-derived used in connection with a stem cell refers to a stem cell which is mobilized from the peripheral blood only, and can include expansion or proliferation of the stem cell in the peripheral blood.
  • the number of circulating stem cells can be increased in the peripheral blood by contacting the peripheral blood with a mobilizing agent, either in vivo or ex vivo, according to the methods as disclosed herein.
  • BM-derived stem cells in the peripheral blood includes stem cells which have proliferated in the bone marrow prior to migration to the peripheral blood, or alternatively stem cells which have proliferated in the peripheral blood after migration from the bone marrow.
  • the number of circulating BM-stem cells can be increased in the peripheral blood by contacting the peripheral blood with a mobilizing agent in vivo according to the methods as disclosed herein.
  • hematopoietic stem cells also referred to as “HSCs,” refers to all stem cells or progenitor cells found inter alia in bone marrow and peripheral blood that are capable of differentiating into any of the specific types of hematopoietic or blood cells, such as erythrocytes, lymphocytes, macrophages and megakaryocytes. HSCs are reactive with certain monoclonal antibodies which are now recognized as being specific for hematopoietic cells, for example, monoclonal antibodies that recognize CD34.
  • hematopoietic cells refers to all types of hematopoietic cells throughout their differentiation from self-renewing hematopoietic stem cells through immature precursor cells of the various blood lineages, including the mature functioning blood cells, as would be understood by persons skilled in the art.
  • mesenchymal cell or “mesenchyme” are used interchangeably herein and refer in some instances to the fusiform or stellate cells found between the ectoderm and endoderm of young embryos. Most mesenchymal cells are derived from established mesodermal layers but in the cephalic region they also develop from neural crest or neural tube ectoderm. Mesenchymal cells have a pluripotential capacity, particularly embryonic mesenchymal cells in the embryonic body, developing at different locations into any of the types of connective or supporting tissues, to smooth muscle, to vascular endothelium, and to blood cells.
  • isolated cell refers to a cell that has been removed from a
  • the cell has been cultured in vitro, e.g., in the presence of other cells.
  • the cell e.g., an isolated target stem cell population produced by the method as disclosed herein, is later introduced into a second subject or re-introduced into the subject from which it (or the cell from which it is descended) was isolated (e.g., allogenic transplantation).
  • isolated population refers to a population of cells that has been removed and separated from a mixed or heterogeneous population of cells.
  • an isolated population is a substantially pure population of cells as compared to the heterogeneous population from which the cells were isolated or enriched from.
  • the isolated population is an isolated population of reprogrammed cells which is a substantially pure population of reprogrammed cells as compared to a heterogeneous population of cells comprising reprogrammed cells and cells from which the reprogrammed cells were derived.
  • substantially pure refers to a population of cells that is at least about 75%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% pure, with respect to the cells making up a total stem cell population.
  • the terms "substantially pure” or “essentially purified,” with regard to a target stem cell population isolated using the methods as disclosed herein, refer to a population of target stem cells that contains fewer than about 20%, more preferably fewer than about 15%, 10%, 8%, 7%, most preferably fewer than about 5%, 4%, 3%, 2%, 1%, or less than 1%, of cells that are a non-target stem cell population as defined by the terms herein.
  • the present invention encompasses methods to expand a target stem cell population, wherein the expanded population of target stem cells is a substantially pure population of target stem cells.
  • proliferating and proliferation refer to an increase in the number of cells in a population (growth) by means of cell division.
  • Cell proliferation is generally understood to result from the coordinated activation of multiple signal transduction pathways in response to the environment, including growth factors and other mitogens.
  • Cell proliferation may also be promoted by release from the actions of intra- or extracellular signals and mechanisms that block or negatively affect cell proliferation.
  • regeneration means regrowth of a cell population, organ or tissue after disease or trauma.
  • reprogrammed cells are capable of renewal of themselves by dividing into the same undifferentiated cells (e.g., pluripotent or non-specialized cell type) over long periods, and/or many months to years.
  • proliferation refers to the expansion of reprogrammed cells by the repeated division of single cells into two identical daughter cells.
  • linear refers to cells with a common ancestry or cells with a common developmental fate, for example, cells that are derived from the same target stem cell population or progeny thereof.
  • clonal cell line refers to a cell lineage that can be maintained in culture and has the potential to propagate indefinitely.
  • a clonal cell line can be a stem cell line (e.g., a target stem cell population cell line) or be derived from a target stem cell population, and where the clonal cell line is used in the context of a clonal cell line comprising a target stem cell population, the term refers to a target stem cell population which has been cultured under in vitro conditions that allow proliferation without differentiation for months to years.
  • Such clonal stem cell lines e.g., a target stem cell population
  • differentiated In the context of cell ontogeny, the adjective “differentiated,” or “differentiating” is a relative term.
  • a “differentiated cell” is a cell that has progressed further down the developmental pathway than the cell it is being compared with.
  • stem cells can differentiate to lineage-restricted precursor cells (such as a mesodermal stem cell), which in turn can differentiate into other types of precursor cells further down the pathway (such as an cardiomyocyte precursor), and then to an end-stage differentiated cell, which plays a characteristic role in a certain tissue type, and may or may not retain the capacity to proliferate further.
  • the term "differentiation" in the present context means the formation of cells expressing markers known to be associated with cells that are more specialized and closer to becoming terminally differentiated cells incapable of further differentiation.
  • the pathway along which cells progress from a less committed cell to a cell that is increasingly committed to a particular cell type, and eventually to a terminally differentiated cell is referred to as progressive differentiation or progressive commitment.
  • Cells which are more specialized e.g., have begun to progress along a path of progressive differentiation
  • partially differentiated are referred to as partially differentiated.
  • Differentiation is a developmental process whereby cells assume a specialized phenotype, e.g., acquire one or more characteristics or functions distinct from other cell types. In some cases, the
  • differentiated phenotype refers to a cell phenotype that is at the mature endpoint in some developmental pathway (a so called terminally differentiated cell). In many, but not all tissues, the process of differentiation is coupled with exit from the cell cycle. In these cases, the terminally differentiated cells lose or greatly restrict their capacity to proliferate.
  • the terms “differentiation” or “differentiated” refer to cells that are more specialized in their fate or function than at a previous point in their development, and includes both cells that are terminally differentiated and cells that, although not terminally differentiated, are more specialized than at a previous point in their development.
  • the development of a cell from an uncommitted cell (for example, a stem cell) to a cell with an increasing degree of commitment to a particular differentiated cell type, and finally to a terminally differentiated cell is known as progressive differentiation or progressive commitment.
  • a cell that is "differentiated” relative to a progenitor cell has one or more phenotypic differences relative to that progenitor cell.
  • Phenotypic differences include, but are not limited to, morphologic differences and differences in gene expression and biological activity, including not only the presence or absence of an expressed marker, but also differences in the amount of a marker and differences in the co-expression patterns of a set of markers.
  • differentiation refers to the cellular development of a cell from a primitive stage towards a more mature (i.e., less primitive) cell.
  • directed differentiation refers to forcing differentiation of a cell from an undifferentiated (e.g., more primitive cell) to a more mature cell type (i.e., less primitive cell) via genetic and/or environmental manipulation.
  • a reprogrammed cell as disclosed herein is subject to directed differentiation into specific cell types, such as neuronal cell types, muscle cell types and the like.
  • the term "media” as referred to herein is a medium for maintaining a tissue or cell
  • the cell culture medium may contain any of the following in an appropriate combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum replacement, and other components such as peptide growth factors, etc.
  • Cell culture media ordinarily used for particular cell types are known to those skilled in the art.
  • phenotype refers to one or a number of total biological characteristics that define the cell or organism under a particular set of environmental conditions and factors, regardless of the actual genotype.
  • a "marker” as used herein describes the characteristics and/or phenotype of a cell.
  • Markers can be used for selection of cells comprising characteristics of interest. Markers will vary with specific cells. Markers are characteristics, whether morphological, functional or biochemical (enzymatic) characteristics particular to a cell type, or molecules expressed by the cell type. Preferably, such markers are proteins, and more preferably, possess an epitope for antibodies or other binding molecules available in the art. However, a marker may consist of any molecule found in a cell including, but not limited to, proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids and steroids. Examples of morphological characteristics or traits include, but are not limited to, shape, size, and nuclear to cytoplasmic ratio.
  • Examples of functional characteristics or traits include, but are not limited to, the ability to adhere to particular substrates, ability to incorporate or exclude particular dyes, ability to migrate under particular conditions, and the ability to differentiate along particular lineages. Markers may be detected by any method available to one of skill in the art.
  • the term "contacting" or “contact” as used herein, as in connection with contacting a peripheral blood sample in vivo, can comprise administering a mobilizing agent, optionally in a composition, to a subject via an appropriate administration route such that the compound contacts the peripheral blood sample in vivo.
  • the term "contacting” or “contact” as used herein, as in connection with contacting a peripheral blood sample ex vivo can comprise administering a mobilizing agent, optionally in a composition, to a peripheral blood sample such that the mobilizing agent contacts the peripheral blood sample ex vivo.
  • administering refers to the placement of a target stem cell population as described herein into a subject by a method or route which results in at least partial localization of the human target stem cell population at a desired site.
  • the human target stem cell population can be administered by any appropriate route which results in delivery to a desired location in the subject where at least a portion of the human target stem cell population remain viable.
  • the period of viability of the cells after administration to a subject can be as short as a few hours, e.g., twenty-four hours, to a few days, to as long as several years.
  • the term "donor” refers to a subject from which an organ, tissue or cell to be transplanted is harvested from.
  • recipient refers to a subject which will receive a transplanted organ, tissue or cell.
  • graft refers to the process whereby a free (unattached) cell, tissue, or organ integrates into a tissue following transplantation into a subject.
  • the term "allograft” refers to a transplanted cell, tissue, or organ derived from a different animal of the same species.
  • xenograft or "xenotransplant” as used herein refer to a transplanted cell, tissue, or organ derived from an animal of a different species.
  • a xenograft is a surgical graft of tissue from one species to an unlike species, genus or family.
  • a graft from a baboon to a human is a xenograft.
  • xenotransplantation refers to the process of transplantation of living cells, tissues or organs from one species to another, such as from pigs to humans.
  • subject and “individual” are used interchangeably herein, and refer to an animal, for example, a human, from whom a target stem cell population as disclosed herein can be isolated and collected according to the methods and compositions described herein, and optionally, a subject can receive a transplantation (e.g., the target stem cell population can be implanted into a subject), for example, for the treatment, including prophylactic treatment, of a disease.
  • a transplantation e.g., the target stem cell population can be implanted into a subject
  • the term “subject” refers to that specific animal.
  • non- human animals and “non-human mammals” are used interchangeably herein, and include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates.
  • subject also encompasses any vertebrate including but not limited to mammals, reptiles, amphibians and fish.
  • the subject is a mammal such as a human, or other mammals such as a domesticated mammal, e.g., dog, cat, horse, and the like, or a production mammal, e.g., cow, sheep, pig, and the like.
  • tissue refers to a group or layer of similarly specialized cells which together perform certain special functions.
  • tissue-specific refers to a source or defining characteristic of cells from a specific tissue.
  • agent means any compound or substance such as, but not limited to, a small molecule, nucleic acid, polypeptide, peptide, drug, ion, etc.
  • An “agent” can be any chemical, entity or moiety, including, without limitation, synthetic and naturally- occurring proteinaceous and non-proteinaceous entities.
  • an agent is nucleic acid, nucleic acid analogues, proteins, antibodies, peptides, aptamers, oligomer of nucleic acids, amino acids, or carbohydrates, including, without limitation, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNAs, lipoproteins, and modifications and combinations thereof etc.
  • agents are small molecule having a chemical moiety.
  • chemical moieties included unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties, including macrolides, leptomycins and related natural products or analogues thereof.
  • Compounds can be known to have a desired activity and/or property, or can be selected from a library of diverse compounds.
  • small molecule refers to a chemical agent which can include, but is not limited to, a peptide, a peptidomimetic, an amino acid, an amino acid analog, a polynucleotide, a polynucleotide analog, an aptamer, a nucleotide, a nucleotide analog, an organic or inorganic compound (e.g., including heterorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.
  • organic or inorganic compound e.g., including heterorganic and organometallic compounds
  • a disease or disorder can also relate to a distemper, ailing, ailment, malady, disorder, sickness, illness, complaint, indisposition or affection.
  • pathology refers to symptoms, for example, structural and functional changes in a cell, tissue, or organs, which contribute to a disease or disorder.
  • the pathology may be associated with a particular nucleic acid sequence, or "pathological nucleic acid” which refers to a nucleic acid sequence that contributes, wholly or in part to the pathology, as an example, the pathological nucleic acid may be a nucleic acid sequence encoding a gene with a particular pathology causing or pathology-associated mutation or polymorphism.
  • the pathology may be associated with the expression of a pathological protein or pathological polypeptide that contributes, wholly or in part, to the pathology associated with a particular disease or disorder.
  • the pathology is, for example, associated with other factors, for example ischemia and the like.
  • treating includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder.
  • parenteral adiBinistration and “administered parenterally” as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal,
  • systemic administration means the administration of a target stem cell population or differentiated progeny thereof and/or their progeny and/or compound and/or other material other than directly into the subject, such that it enters the animal's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous or intravenous administration.
  • phrases "pharmaceutically acceptable carrier” as used herein means a
  • composition or vehicle such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body.
  • a liquid or solid filler such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
  • drug refers to a chemical entity or biological product, or combination of chemical entities or biological products, administered to a subject to treat or prevent or control a disease or condition.
  • the chemical entity or biological product is preferably, but not necessarily a low molecular weight compound, but may also be a larger compound, for example, an oligomer of nucleic acids, amino acids, or carbohydrates including without limitation proteins, oligonucleotides, ribozymes, DNAzymes,
  • glycoproteins glycoproteins, siR As, lipoproteins, aptamers, and modifications and combinations thereof.
  • transplantation refers to introduction of new cells (e.g., a target stem cell population or differentiated progeny thereof), or tissues (such as differentiated cells produced from a target stem cell population), or organs into a host (i.e., transplant recipient or transplant subject).
  • modulate is used consistently with its use in the art, e.g., meaning to cause or facilitate a qualitative or quantitative change, alteration, or modification in a process, pathway, or phenomenon of interest. Without limitation, such change may be an increase, decrease, or change in relative strength or activity of different components or branches of the process, pathway, or phenomenon.
  • a “modulator” is an agent that causes or facilitates a qualitative or quantitative change, alteration, or modification in a process, pathway, or phenomenon of interest.
  • the terms “decrease” , “reduced,” “reduction,” “decrease” or “inhibit” are all used herein generally to mean a decrease by a statistically significant amount. However, for avoidance of doubt, “reduced,” “reduction” or “decrease” or “inhibit” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.
  • a 100% decrease e.g., absent level as compared to a reference sample
  • the terms "increased,” “increase” or “enhance” or “activate” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • the term "statistically significant” or “significantly” refers to statistical significance and generally means a two standard deviation (2SD) below normal, or lower, concentration of the marker.
  • the term refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. The decision is often made using the p-value.
  • substantially as used herein means a proportion of at least about 60%, or preferably at least about 70%, or at least about 80%, or at least about 90%, at least about 95%, at least about 97%, or at least about 99% or more, or any integer between 70% and 100%.
  • One aspect of the present invention is to separate different target stem cell populations from the peripheral blood into subpopulations of stem cells using centrifugal elutriation.
  • a peripheral blood sample is introduced into a generally funnel-shaped separation chamber located on a spinning centrifuge.
  • a flow of liquid elutriation buffer or low density liquid is then introduced into the chamber containing the peripheral blood sample.
  • the liquid sweeps smaller sized, slower-sedimenting cells toward an elutriation boundary within the chamber, while larger, faster-sedimenting cells migrate to an area of the chamber where the centrifugal force and the sedimentation (drag) forces are balanced.
  • the peripheral blood e.g., mobilized peripheral blood
  • one aspect of the present invention relates to the separation of these different stem cell populations present in peripheral blood into distinct populations using elutriation, where smaller stem cells are fractionated from stem cells which are larger in size.
  • any elutriation device can be used to fractionate a peripheral blood sample into fractions comprising different stem cell populations.
  • commercially available elutriation devices can be used, for example, the ELUTRA® centrifuge manufactured by Gambro BCT, Inc. The ELUTRA CELL
  • the ELUTRA CELL SEPARATION SYSTEM® enables the separation of cell populations into multiple fractions based on both size and density, enabling cell enrichment, depletion, concentration, and washing all within a functionally-closed system.
  • the ELUTRA CELL SEPARATION SYSTEM® enables enrichment of stem cell populations directly from leukapheresis products without antibodies or preprocessing in less than one hour.
  • SEPARATION SYSTEM® uses counterflow centrifugal elutriation, where fluid flows through cell layers in a centrifugal field in order to separate cell populations.
  • other elutriation devices can be used, including but not limited to, the COBE® Spectra apheresis system, the TRIMA® system and the TRIMA ACCEL® system, also manufactured by Gambro BTC Inc., as well as other commercially available elutriation devices used to separate blood components.
  • the peripheral blood is fractionated using a cell separator such as the COBE® Spectra Apheresis System.
  • a cell separator such as the COBE® Spectra Apheresis System.
  • the COBE® SPECTRATM centrifuge is described in U.S. Patents 4,425,172; 4,708,712; and 6,022,306, which are incorporated herein by reference.
  • the peripheral blood sample is drawn into a cell separator such as the COBE® Spectra Apheresis System, and, optionally, an anticoagulant solution is added to the blood to keep it from clotting during the procedure.
  • the blood/anticoagulant admixture cycles through a centrifuge to separate the peripheral blood sample into stem cell populations and mononuclear cells from the other blood components and plasma.
  • the system pumps the separated stem cells into a collection bag for storage, while the other blood components and plasma return to the patient. All tubing sets and needles used are sterile, so there is no risk of disease transmission.
  • leukapheresis refers to one type of separation procedure which separates white blood cells from the other particles, and typically uses a centrifuge. The resulting harvested or separated white blood cells can then be further separated into subsets of desired cells (e.g., monocytes, lymphocytes, and granulocytes) for collection or culture, if desired, although it is understood that collection of other cells may also be desired.
  • desired cells e.g., monocytes, lymphocytes, and granulocytes
  • the collected leukapheresis products are often contaminated with platelets and red blood cells which can interfere with various cell separation and/or cell selection techniques and later cultivation of the selected cells for therapeutic use. Thus, it is desirable to separate the white blood cells into the desired subsets and remove or reduce the number of platelets and red blood cells.
  • elutriation a method for the separation or fractionation of white blood cells from other particles and into more purified selected subsets.
  • One such method is centrifugal elutriation.
  • a cell batch is introduced into a generally funnel-shaped separation chamber located on a spinning centrifuge.
  • a flow of liquid elutriation buffer or low density liquid is then introduced into the chamber containing the cell batch.
  • the liquid sweeps smaller sized, slower- sedimenting cells toward an elutriation boundary within the chamber, while larger, faster- sedimenting cells migrate to an area of the chamber where the centrifugal force and the sedimentation (drag) forces are balanced.
  • the eluuiation method as used herein can separate the peripheral blood sample into various target stem cell populations which does not affect the function or viability of the stem cells.
  • Collection may be performed from peripheral blood obtained from any subject, such as a human subject, including an adult or a non-neonate child. Furthermore, collection may involve one or more collecting steps or collecting periods. For example, collection (e.g., using an apheresis process) may be performed at least two times, at least three times, or at least 5 times, on a person.
  • the number of total nucleated cells collected per kilogram weight of the person may be one million (lxlO 6 ) or more (e.g., 1 xlO ', 1 xlO 8 , lxlO 9 , lxlO 10 , lxlO 11 , lxlO 12 , lxlO 13 lxlO 14 , lxlO 15 , lxlO 16 , lxlO 17 , lxlO 18 , lxlO 19 , lxl
  • the number of cells collected in a single collection session may be equal or greater than 1x10 13 , total nucleated cells, or at least on the order of lxlO 14 , lxlO 13 , lxlO 12 , lxlO 11 , lxlO 10 , lxlO 9 , lxlO 8 , lxlO 7 , lxlO 6 ) or more (
  • stem cell populations can be collected when the subject is within a weight range from 10 to 200 kg in accordance with one embodiment of the present invention, or any range within such range, such as 20 to 40 kg.
  • the subject may be of a certain age, within a range from 2-80 years old (e.g., 2-10, 10-15, 12-18, 16-20, 20-26, 26-30, 30-35, 30- 40, 40-45, 40-50, 55-60, 60-65, 60-70, and 70-80 years old) in accordance with one embodiment of the present invention.
  • 2-80 years old e.g., 2-10, 10-15, 12-18, 16-20, 20-26, 26-30, 30-35, 30- 40, 40-45, 40-50, 55-60, 60-65, 60-70, and 70-80 years old
  • the rate of the flow of the liquid elutriation buffer determines the size of the target stem cells to be separated from the remaining stem cells.
  • a target stem cell population of small stem cells for example, including, but not limited to, VSELs can be separated from the other stem cells in the peripheral blood when the elutriation flow rate is at least about 50ml/minute.
  • a target stem cell population of stem cells which are larger than the size of VSELs can be separated from the other stem cells in the peripheral blood when the elutriation flow rate is at least about 70ml/minute.
  • a target stem cell population of stem cells can be separated from the other stem cells in the peripheral blood when the elutriation flow rate is at least about 90ml/minute. In some embodiments, a target stem cell population of stem cells can be separated from the other stem cells in the peripheral blood when the elutriation flow rate is at least about >90ml/minute, or about 105ml/minute. [00189] In some embodiments, a flow rate of less than about 20ml/mmute can be used to collect platelets from the peripheral blood. In some embodiments, a flow rate of between about 2ml/minute to about 20ml/minute can be used to collect platelets.
  • the first fraction is collected at a flow rate of about 20ml/minute or less.
  • a second fraction is collected at a flow rate of between 20ml/min to about 50ml/min, such as about at least 20ml/minute, or at least 30ml/minute, or about 40ml, minute, or about 50ml/minute.
  • a third fraction is collected at a flow rate of between 51ml/min to about 70ml/min, such as about at least 51 ml/minute, or at least 60ml/minute, or about 65ml, minute, or about 70ml/minute.
  • a fourth fraction is collected at a flow rate of between 71ml/minute to about 90ml/minute, such as about at least 71ml/minute, or at least 80ml/minute, or about 85ml/minute, or about 90ml/minute.
  • a fifth fraction is collected at a flow rate of between about 91m1 ⁇ 2ninute to about 105ml/minute or greater than 105ml/minute, such as about at least 91ml/minute, or at least 95ml/minute, or about lOOml/minute, or about 105ml/minute.
  • the fifth fraction can be collected as subtractions, for example cells can be collected at one or more of the following flow rates; at least about 95ml/minute, or at least about 1 OOml'minute, or about 105ml/minute, or greater than
  • Such an embodiment may be useful to collect subtractions comprising substantially different target stem cell populations, for example, where one subtraction of collected cells comprises substantially HSCs, and another subtraction of collected cells comprises substantially MSC.
  • the elutriation flow r rate is set or other parameters are set to allow separated fractions to be continuously analyzed at the time of the separation process not only with respect to cell size but also in relation to other cell properties of importance, such as specific density, morphology, and cell surface markers, as disclosed in U.S. Patent No.
  • optimal setting refers to the setting which leads to such fractionation that, in the cell population of a fraction, an optimal number of cells are present with a cell property of a particular value and lying within a particular range of values.
  • optimal settings for fractionation of a peripheral blood sample such as a mobilized peripheral blood sample, are shown in Table 1.
  • Table 1 optimal settings for separating different target stem cell populations from
  • the desired target stem cell population in a fraction may be less than 1% of the total number of cells in the fraction obtained by the elutriation separation process, e.g., as in the case of Fraction 2, where the desired target population is VSELs (Very Small Embryonic-like cells) which comprise 0.3% of the total cells in Fraction 2, but is a 30-fold enrichment as compared to the pre-elutriation peripheral blood sample.
  • VSELs Very Small Embryonic-like cells
  • the second fraction (Fraction 2), or the fraction collected from a flow rate of about 50ml/minute comprises at least about 0.05%, or at least about 0.1%, or at least about 0.2%, or about 0.3%, or at least about 0.4%, or greater than 0.4% of VSELs.
  • the second fraction (Fraction 2), or a fraction of cells collected from a flow rate of about 50ml/minute can be separated into different subpopulations of cells, for example, into a substantially pure population of VSELs.
  • a substantially pure population of VSELs comprise at least about 75%, preferably at least about 85%, more preferably at least about 90 %, and most preferably at least about 95% VSELs, with respect to the cells making up a total cell population.
  • the fifth fraction (Fraction 5), or the fraction collected from a flow rate of greater than 90ml/minute, such as about 105ml/minute or greater than 105ml/minute comprises at least about 1%, or at least about 2%, or at least about 3%, or about 4% or at least about 5%, or greater than 5% of a cells which are either HSCs or MSCs.
  • the fifth fraction (Fraction 5), or a fraction of cells collected from a flow rate of about 105ml/minute or greater than 105ml/minute can be further fractionated such that the population of HSCs and MSCs can be separated from non-HSC cells and non-MSC cells, and, further, the HSC cells can be separated from a population of MSCs. Accordingly, the fifth fraction (Fraction 5), or a fraction of cells collected from a flow rate of about
  • >90ml/minute, such as 105ml/minute, or greater than 105ml/minute can be separated into subpopulations of cells, for example, into a substantially pure population of HSC cells and into a substantially pure population of MSC cells.
  • a substantially pure population of HSCs comprises at least about 75%, preferably at least about 85 %. more preferably at least about 90%, and most preferably at least about 95% HSCs, with respect to the cells making up a total cell population.
  • a substantially pure population of MSCs comprise at least about 75%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% MSCs, with respect to the cells making up a total cell population
  • the fractionation of the peripheral blood into fractions is not limited in number.
  • the peripheral blood can be subjected to any number of different flow rates to yield any number of fractions.
  • the peripheral blood can be separated into at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 11 , or at least 12, or more than 12 different fractions.
  • the peripheral blood can be separated into a number multiple different fractions by subjecting the peripheral blood to at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10, or at least 1 1, or at least 12, or more than 12 different flow rates.
  • the desired yield of a target stem cell population is determined by the target collected volume as follows:
  • yield of target stem cells target collected volume for the target stem cell population
  • the target flow rate can be calculated as discussed in Table 1 in U.S. Patent No.
  • the centrifugal elutriation separates cell particles having different sedimentation velocities, as disclosed in U.S. Patent No. 7,201,848, which is incorporated herein in its entirety by reference.
  • Stoke 's law describes sedimentation velocity (SV) of a spherical particle, as follows:
  • r is the radius of the particle
  • pp is the density of the particle
  • p m is the density of the liquid medium
  • ⁇ is the viscosity of the medium
  • g is the gravitational or centrifugal acceleration.
  • centrifugal elutriation has a number of limitations. In most of these processes, particles must be introduced within a flow of fluid medium in separate, discontinuous batches to allow for sufficient particle separation. Thus, some elutriation processes only permit separation in particle batches and require an additional fluid medium to transport particles. In addition, flow forces must be precisely balanced against centrifugal force to allow for proper particle segregation. Thus, the present invention encompasses centrifugal elutriation as disclosed in U.S. Patent Application 2008/0318756, which is incorporated herein in its entirety by reference.
  • Peripheral blood sample for enriching subpopulations of stem cells by elutriation
  • the peripheral blood sample which is used for the methods as disclosed herein is obtained from a mammalian subject, such as a human subject.
  • the human subject has been previously administered a mobilizing agent to increase the yield or number of circulating stem cells in the peripheral blood.
  • Mobilization methods are known in the art, and include without limitation, methods as disclosed in U.S. Patent No. 6,261,549 and U.S. Patent Application 2009/0155225, which are incorporated herein by reference in their entirety.
  • mobilizing agents administered to a subject include, but are not limited to, G-CSF, GM-CSF, dexamethasone, a CXCR4 receptors inhibitor, Inter leukin- 1 (IL-1), Interleukin-3 (IL-3), Interleukin-8 (IL-8), PIXY-321 (GM-CSF/IL-3 fusion protein), macrophage inflammatory protein, stem cell factor, thrombopoietin and growth related oncogene, as single mobilization agents or in any combination of such mobilizing agents.
  • mobilizing agents include for example, but are not limited to CXCR4 inhibitors, such as AMD3100, ALX40-4C, T22, T134, T140 and TAK-779, which are disclosed in U.S. Patent No. 7,169,750, which is incorporated herein by reference in its entirety.
  • CXCR4 inhibitors such as AMD3100, ALX40-4C, T22, T134, T140 and TAK-779, which are disclosed in U.S. Patent No. 7,169,750, which is incorporated herein by reference in its entirety.
  • the peripheral blood sample is a mobilized peripheral blood
  • the peripheral blood is harvested or obtained from a subject after at least 2 days following administration of a mobilizing agent. In some embodiments, the peripheral blood sample is harvested or obtained from a subject after 4 days or less following administration of an effective amount of a mobilizing agent to the subject.
  • a peripheral blood sample or mobilized peripheral blood sample is pre-treated, e.g., by apheresis or some other method (e.g., chemical lysis of lymphocytes) prior to elutriation process to separate multiple target stem cell populations according to the methods as disclosed herein.
  • the starting material for elutriation according to the methods as disclosed herein is an apheresis product rather than the peripheral blood sample itself, where the apheresis product is processed from the peripheral blood sample.
  • the subjects amenable to collecting a variety of stem cell populations from peripheral blood using the methods as disclosed herein can by any subject, such as any human subject, at any age.
  • the subject is a child, such as from age of about 1 to 18 years of age.
  • the subject is an adult subject.
  • the subject is a healthy subject.
  • the subject has a predisposition to a disease (e.g., diabetes, cardiovascular disease, etc.), such as a subject having a mutation or genetic marker (such as a SNP, etc.) in a gene which is associated to an increase risk that the subject will likely develop the disease at some future date.
  • a disease e.g., diabetes, cardiovascular disease, etc.
  • a subject having a mutation or genetic marker such as a SNP, etc.
  • the subject is administered a mobilizing agent prior to obtaining a peripheral blood sample in the subject.
  • the subject is preferably at least 10 years old, or at least 18 years old, or at least about 30 years old.
  • the subject is preferably less than about 60 years of age, or less than about 50 years of age, or less than about 40 years of age, or less than about 30 years of age, or less than about 20 years of age.
  • the hematocrit range is selected to enhance the proportion of VSELs.
  • the hematocrit range is selected to enhance cryopreservation of collected VSELs, for example by minimizing collection of granulocytes.
  • the hematocrit range is selected to ensure simultaneous collection of multiple stem cell populations, such as, for example, VSELs and MSCs.
  • the hematocrit range is selected to be 2-3%. In another embodiment, the hematocrit range is selected to be 3-4%
  • the enriched populations of target stem cells are stored for future use in a stem cell bank, e.g., for use in the treatment of a disease or protocol for regenerative medicine or therapy for the subject of whom the cells were originally obtained (e.g., the cells are used in subsequent allogenic transplantation procedures).
  • the target stem cells obtained by the methods as disclosed herein are processed for moderate to long-term storage in a cell bank. This procedure has advantages in that different target stem cell populations can be stored such that the target stem cells population most suitable for the subsequent procedure can be selected for use in the regenerative therapy procedure.
  • the present invention provides the ability to tailor the use of the target stem cell populations for future use.
  • the different populations of stem cells obtained from peripheral blood by the methods as disclosed herein can be cryopreserved (e.g., frozen) at liquid nitrogen temperatures and stored for long periods of time, being capable of use on thawing, for example, for use in therapeutic purposes such as regenerative therapy or medicine.
  • the fractions obtained from the peripheral blood can be further separated into subpopulations of stem cells prior to cryopreservation.
  • the different target populations of stem cells obtamed in each fraction can be further purified and expanded (e.g., propagated by in vitro culturing) to generate a substantially pure population of the target stem cells prior to being cryopreserved .
  • the stem cell populations can be stored in a 10% DMSO, 50% FCS, 40% RPMI 1640 medium. Once thawed, the stem cell populations can be cultured and expanded ex vivo in the presence of growth factors and/or feeder layers for an appropriate period of time prior to implanting into a subject in need thereof.
  • each population of stem cells obtained from peripheral blood by the methods as disclosed herein can be suspended in DPBS and may be placed on ice for at least about 15 minutes in preparation for cryopreservation.
  • the preparation may comprise adding cryopreservation media to the target stem cell population or a substantially pure population of target stem cells and then subjecting the mixture to several temperature reduction steps to reduce the temperature of the target stem cell population or a substantially pure population of target stem cells to a final temperature of about -90° C, utilizing a controlled rate freezer or other suitable freezer system (dump-freeze monitored or a freeze container (Nalgene)).
  • Suitable control rate freezers include, but are not limited to, Cryomed Thermo Form a Controlled Rate Freezer 7454 (Thermo Electron, Corp.), Planar Controlled Rate Freezer Kryo 10/16 (TS Scientific), Gordinier, Bio-Cool-FTS Systems, and Asymptote EF600, BIOSTOR CBS 2100 series.
  • Cryopreservation media may be prepared comprising media and DMSO.
  • About 3 ml of DPBS may be added to a container, such as, for example, a 50 ml conical tube.
  • About 1 ml of human serum albumin (HSA) may be added to the about 3 ml of DPBS and then chilled for about ten minutes on ice.
  • About 1 ml of the chilled 99% DMSO is added to the HSA and DPBS to prepare the final cryopreservation media.
  • Cryopreservation media and the target stem cell population, or a substantially pure population of target stem cells may then be placed on ice for about 15 minutes before the cryopreservation media is added to the cell sample.
  • Batch processing may be used for aliquoting cryopreservation media into a cell sample.
  • a single aliquot of about 100 ⁇ of the target stem cell population, or a substantially pure population of target stem cells may be combined with about 3 ml of DPBS, 1 ml of HSA, and about 1 ml of 99% DMSO.
  • About 2 aliquots of about 200 ⁇ of MSC cell suspension may be combined with about 6 ml of DPBS, 2 ml of HSA, and about 2 ml of 99% DMSO.
  • About 5 aliquots of cell sample may be combined with about 15 ml of DPBS, about 5 ml of HSA, and about 5 ml of 99% DMSO.
  • About 10 aliquots of cell sample may be combined with about 30 ml of DPBS, about 10 ml of HSA, and about 10 ml of 99% DMSO.
  • cryopreservation media with cryopreservation agents may be used to maintain a high cell viability outcome post-thaw, such as, for example, CryoStor CS10 or CS5 (Biolife), embryonic cryopreservation media supplemented with propanediol and sucrose (Vitrolife), or SAGE media (Cooper Surgical).
  • Glycerol may be used with other cryopreservation agents, such as, DMSO, or may be used alone at a concentration of about 10% in a media with suitable protein.
  • cryopreservation media may be added to the target stem cell population, or a substantially pure population of target stem cells.
  • Cryopreservation media may be added to the target stem cell population, or a substantially pure population of target stem cells suspended in DPBS drop by drop until the volume of total mixture of suspended menstrual stem cells and cryopreservation media is brought to the final desired volume of cryopreservation cell mixture.
  • the final cryopreservation cell media may be transferred into 2 ⁇ 5 ml bar-coded cryoquats with the QC sample stored in the cap of the 5 ml vial. Use a pipettor to remove about 200 ⁇ aliquots of the sample and aliquot into the QC caps.
  • cryovials should be placed in the CRYOMED® Freezer and subject to temperature reduction by controlled rate to a temperature at or below about -85° C.
  • the cryopreserved specimens should be transferred to a bunker for storage in the vapor of Liquid Nitrogen at -150° C. or less. Any sample that tests positive for an infectious disease should be quarantined. Any sample that tests negative for infectious disease may be transferred to a different permanent storage.
  • the following temperature reduction steps may be programmed in the controlled rate freezer, first reducing the mixture of cryopreservation agent and the target stem cell population, or a substantially pure population of target stem cells.
  • cryopreservation agent may be subjected to controlled rate temperature reductions in preparation for final storage in a freezer.
  • the controlled rate reductions may be designed to maintain cell viability.
  • a Cryo-Med Freezer (Thermo Electron Corp.), liquid nitrogen cylinder, and portable Cryo-Med Freezer may be used for controlled rate reductions in preparation for final storage in a freezer.
  • the cells may be subject to controlled rate reductions in cryovials or ciyobags to reach a temperature of about -90° C.
  • the cells may be subject to the following controlled rate reduction profile: wait at about 4° C, 1.0° C. per minute to -6.0° C. (sample), 25.0° C. per minute to -50.0° C. (chamber), 10.0° C. per minute to -14.0° C. (chamber), 1.0° C. per minute to -45.0° C. (chamber), 10.0° C. per minute to -90.0° C. (chamber), and end (sample at or below -85.0° C).
  • the cells may be subject to the following controlled rate reduction profile: wait at 4.0° C, 1.0° C. per minute to -3.0° C. (chamber), 10.0° C. per minute to -20.0° C. (chamber), 1.0° C. per minute to -40.0° C. (chamber), 10.0° C. per minute to -90.0° C. (chamber), and end.
  • cryopreservation vials are transferred to a cryogenic storage unit and stored in the vapor of liquid Nitrogen at a temperature at or below about -135° C. or alternatively vials may be stored in the liquid phase of liquid nitrogen.
  • a suitable cryogenic storage unit includes, but is not limited to, LN2 Freezer MVE 1830 (Chart Industries).
  • Fresh samples of the target stem cell population, or a substantially pure population of target stem cells and thawed samples of the target stem cell population, or a substantially pure population of target stem cells that were previously ciyopreserved, may also be analyzed by flow cytomeuy to analyze cell surface markers, cell viability, and other cell characteristics.
  • a sample of a fresh sample of the target stem cell population, or a substantially pure population of target stem cells may also be analyzed following cell lysis, e.g. using immunoblot analysis and the like.
  • the ciyopreserved target stem cell population, or a substantially pure population of target stem cells may be thawed according to the thawing process described herein. In cases where the target stem cell population, or a substantially pure population of target stem cells, must be thawed, the target stem cell population, or a substantially pure population of target stem cells, may require flow cytometry analysis immediately after thaw or after cells are cultured to assess a certain cell passage.
  • the ciyopreserved samples may be agitated in a 37° C. water bath without letting the cells completely thaw.
  • the cells may be transferred into about 1 ml of chilled wash media and mixed by inversion.
  • the sample may be centrifuged at about 2000 rpm for about seven minutes. The supernatant may be removed and the cells resuspended in about 100 ⁇ of wash media (25% HSA, DNAse, Heparin and HBSS w/Ca ++ and Mg ⁇ ).
  • the resuspended cells may then be centrifuged in a Blood Bank Serofuge for about 1 minute. The supernatant may be decanted and the cells resuspended in about 1.2 ml Sheath fluid and vortexed.
  • the target stem cell population, or a substantially pure population of target stem cells obtained by the methods as disclosed herein, such as moderate or long-term storage in a cell bank.
  • moderate to long term storage of cells in a cell bank is disclosed in U.S. Patent Application No. 2003/0054331 and International Patent Publication No. WO 03/024215, which are incorporated by reference in their entireties.
  • the target stem cell population, or a substantially pure population of target stem cells as disclosed herein may be loaded into a delivery device, such as a syringe, for placement into the recipient subject by any means known to one of ordinary skill in the art.
  • a target stem cell population obtained using the methods as disclosed herein can be suspended in a short term storage medium suitable for infusion into a subject, such as the media disclosed in U.S. Patent No. 5,955,257, which is incorporated herein in its entirety by reference.
  • the target stem cell populations enriched using the methods as disclosed herein can be stored (e.g., for future therapeutic use by the donor (e.g., allogenic) or by another subject) or directly transplanted, e.g., for use to promote postsurgical healing.
  • the plurality of stem cell populations collected by the methods as disclosed herein can be further sorted into at least two subpopulations which may be cryopreserved separately or together (e.g., in the same vial).
  • at least two subpopulations of stem cells may be two subpopulations of the stem cells.
  • Fraction 2 can be sorted into at least two subpopulations of cells, for example, but not limited to (1) a stem cell population or a population enriched for VSELs and (2) a non- stem cell population or a population depleted for VSELs.
  • Fraction 5 can be sorted into at least two subpopulations of cells, for example, but not limited to, (1) a stem cell population of HSCs or a population enriched for HSCs and (2) a stem cell population of MSCs or a population enriched for MSCs, and (3) a non stem cell population or a population depleted for HSCs or MSCs.
  • the two subpopulations i.e., (1) and (2) above
  • population (3) may be cryopreserved together.
  • this sorting of stem cells of a fraction into subpopulations can be by positive or negative sorting, or a combination thereof.
  • markers of cell surface proteins can be used to positively select for or negatively select or remove stem cells. Examples of such markers are well known in the art, and are shown in Table 2.
  • HSCs Hematopoietic stem cells
  • MSCs Mesenchymal stem cells
  • a target stem cell population, or a substantially pure population of target stem cells obtained by the methods as disclosed herein can optionally be packaged in a suitable container, in the presence of suitable media.
  • the package further comprises written instructions for a desired purpose, such as methods of implantation into a subject (and optionally methods of storage and/or methods of thawing if
  • cryopreserved of the target stem cell population, or a substantially pure population of target stem cells for the improvement or treatment of a disease, or for a treatment protocol for regenerative medicine or therapy.
  • the target stem cell populations collected from the peripheral blood of a subject by the methods as disclosed herein can be introduced or transplanted back to the individual when the subject is in need of such cellular therapy.
  • a stem cell composition comprising a target stem cell population collected from the peripheral blood of a subject by the methods as disclosed herein can be used to repair, treat, or ameliorate various aesthetic or functional conditions (e.g., defects) through the augmentation of damaged tissues.
  • the target stem cell populations collected from the peripheral blood of a subject by the methods as disclosed herein provide an important resource for rebuilding or augmenting damaged tissues, and thus represent the ability to collect medically useful multiple stem cell populations from a subject from a single source.
  • the target stem cell populations collected from the peripheral blood of a subject by the methods as disclosed herein can be used in tissue engineering and regenerative medicine for the replacement of body parts that have been damaged by developmental defects, injury, disease, or the wear and tear of aging.
  • the target stem cell populations collected from the peripheral blood of a subject by the methods as disclosed herein provide a unique system in which the multiple different stem cell populations can be differentiated into different cells and give rise to specific lineages of the same individual or genotypes.
  • the target stem cell populations collected from the peripheral blood of a subject by the methods as disclosed herein therefore provide significant advantages for
  • target stem cell populations collected from the peripheral blood of a subject by the methods as disclosed herein and compositions thereof can be used for augmenting soft tissue not associated with injury by adding bulk to a soft tissue area, opening, depression, or void in the absence of disease or trauma, such as for "smoothing.”
  • Multiple and successive administrations of target stem cell populations collected from the peripheral blood of a subject by the methods as disclosed herein are also embraced by the present invention.
  • peripheral blood of a subject by the methods as disclosed herein are preferably collected from an autologous or heterologous human or animal source.
  • An autologous animal or human source is more preferred.
  • Stem cell compositions are then prepared and isolated as described herein.
  • a suspension of mononucleated cells is prepared. Such suspensions contain concentrations of a target stem cell population collected from the peripheral blood of a subject by the methods as disclosed herein in a physiologically-acceptable carrier, excipient, or diluent.
  • stem cell suspensions may be in serum-free, sterile solutions, such as cryopreservation solutions. Enriched stem cell preparations may also be used. The stem cell suspensions may then be introduced, e.g., via injection, into one or more sites of the donor tissue.
  • Concentrated or enriched stem cells may be administered as a pharmaceutically or
  • physiologically acceptable preparation or composition containing a physiologically acceptable carrier, excipient, or diluent, and administered to the tissues of the recipient organism of interest, including humans and non-human animals.
  • the stem cell-containing composition may be prepared by resuspending the cells in a suitable liquid or solution such as sterile physiological saline or other physiologically acceptable injectable aqueous liquids.
  • suitable liquid or solution such as sterile physiological saline or other physiologically acceptable injectable aqueous liquids.
  • the amounts of the components to be used in such compositions can be routinely determined by those having skill in the art.
  • the target stem cell populations collected from the peripheral blood of a subject by the methods as disclosed herein or compositions thereof may be administered by placement of the stem cell suspensions onto absorbent or adherent material, e.g., a collagen sponge matrix, and insertion of the stem cell-containing material into or onto the site of interest.
  • the target stem cell populations collected from the peripheral blood of a subject by the methods as disclosed herein may be administered by parenteral routes of injection, including subcutaneous, intravenous, intramuscular and intrasternal. Other modes of administration include, but are not limited to, intranasal, intrathecal, intracutaneous, percutaneous, enteral, and sublingual.
  • a target stem cell population collected from the peripheral blood of a subject by the methods as disclosed can be administered by endoscopic surgery.
  • a composition comprising a target stem cell population collected from the peripheral blood of a subject by the methods as disclosed herein can be suspended in a sterile solution or suspension or may be resuspended in pharmaceutically- and physiologically-acceptable aqueous or oleaginous vehicles, which may contain preservatives, stabilizers, and material for rendering the solution or suspension isotonic with body fluids (i.e., blood) of the recipient.
  • excipients suitable for use include water, phosphate buffered saline, pH 7.4, 0.15 M aqueous sodium chloride solution, dextrose, glycerol, dilute ethanol, and the like, and mixtures thereof.
  • Illustrative stabilizers are polyethylene glycol, proteins, saccharides, amino acids, inorganic acids, and organic acids, which may be used either on their own or as admixtures.
  • the amounts or quantities, as well as the routes of administration used, are determined on an individual basis, and correspond to the amounts used in similar types of applications or indications known to those of skill in the art.
  • a population of target stem cells collected from the peripheral blood of a subject by the methods as disclosed herein can be administered to body tissues, including epithelial tissue (e.g., skin, lumen, etc.) muscle tissue (e.g., smooth muscle), blood, brain, and various organ tissues such as those organs that are associated with the urological system (e.g., bladder, urethra, ureter, kidneys, etc.).
  • a target stem cell population collected from the peripheral blood of a subject by the methods as disclosed herein can comprise other cells, (e.g., a fraction of cells comprising a target stem cell population) and can be administered to a subject, for example, by infusion into the blood stream of a subject through an intravenous (i.v.) catheter, like any other i.v. fluid.
  • an individualized mixture of cells may be generated so as to provide a cellular therapy mixture specific for therapeutic needs of a subject.
  • the comprehensive mixture of cells obtained such as through an apheresis process may be characterized, sorted, and segregated into distinct cell populations.
  • Cell markers such as VSELs, MSG and HSC markers or tissue specific markers may be used to phenotypically characterize the populations of cells collected from the peripheral blood. Using these markers, it is possible to segregate and sort on the basis of cell type.
  • the mixture of cells is thus transformed into populations of cells, which may be broadly classified into two portions: a stem cell portion and a non-stem cell portion.
  • the non-stem cell portion may further be classified into a progenitor cell or fibroblast portion and a functional cell or fully differentiated cell portion.
  • the stem cell and non-stem cell portions may be cryopreserved and stored separately- In this manner, a library or repository of distinct cell populations from a subject may be created.
  • stem cell and non-stem cell portions may the cryopreserved together and then sorted and separated prior to use.
  • the target stem cell populations collected from the peripheral blood of a subject by the methods as disclosed herein can be used to generate or differentiate into any population of a cell type that developed from a germ layer (i.e., endoderm, mesoderm, and ectoderm).
  • differentiated cells include, but are not limited to, differentiated cells, neural progenitor or differentiated cells, glial progenitor or differentiated cells, oligodendrocyte progenitor or differentiated cells, skin progenitor or differentiated cells, hepatic progenitor or differentiated cells, muscle progenitor or differentiated cells, bone progenitor or differentiated cells, mesenchymal stem or progenitor cells, pancreatic progenitor or differentiated cells, progenitor or differentiated chondrocytes, stromal progenitor or differentiated cells, cultured expanded stem or progenitor cells, cultured differentiated stem or progenitor cells, or combinations thereof.
  • hematopoietic cells which may include any of the nucleated cells which may be involved with the erythroid, lymphoid or
  • myelomonocytic lineages as well as myoblasts and fibroblasts.
  • progenitor cells such as hematopoietic, neural, stromal, muscle (including smooth muscle), hepatic, pulmonary, gastrointestinal, and mesenchymal progenitor cells.
  • differentiated cells such as, osteoblasts, hepatocytes, granulocytes, chondrocytes, myocytes, adipocytes, neuronal cells, pancreatic, or combinations and mixtures thereof.
  • a target stem cell population collected from the peripheral blood of a subject by the methods as disclosed herein can be combined, recombined, or compounded into a cellular therapy mixture of cells appropriate for treating the disease of a subject and/or regenerating a specific tissue.
  • a combination of target stem cell populations collected from the peripheral blood of a subject by the methods as disclosed herein, tissue specific progenitor cells, and optionally functional cells can be used, for example, to enhance the engraftment of the transplanted stem cells.
  • a cellular therapy product may comprise: from about 10% to about 90% of a specific target stem cell population collected from the peripheral blood of a subject by the methods as disclosed herein, about 10% to about 80%, or about 10% to about 60%, or about 10% to about 40%, or about 10% to about 90%, of a target stem cell population collected from the peripheral blood of a subject by the methods as disclosed herein.
  • a target stem cell population can also comprise a non-stem cell population, such as about from about 5% to about 50% functional cells, about 5% to about 40% functional cells, about 5% to about 30% functional cells, about 5% to about 20% functional cells, or about 5% to about 10% functional cells.
  • the isolated target stem cell populations provide a library of different stem cell types from a particular individual which can be maintained in culture and/or cryopreserved for future use, e.g., for use alone, or selectively recombined (e.g., custom mixing) for individualized autologous therapeutic applications in regenerative therapy.
  • a suitable example of the cellular therapy product described above is the autologous mixture of HSCs and MSCs or other custom mixing of autologous stem cell populations, or with other functional cells of the hematopoietic system.
  • Another example is a cellular therapy product comprising an autologous mixture of PBSCs, myocardial progenitor cells, and optionally myocardial cells.
  • a method of treating a patient in need thereof comprising administering to a subject an autologous mixture of one or more target stem cell populations, either alone or in combination (either separately or in an admixture) collected from the peripheral blood of a subject by the methods as disclosed herein.
  • a target stem cell populations collected from the peripheral blood of a subject by the methods as disclosed herein can be stored in a cell bank to support an elective healthcare insurance model to effectively protect members of the population from future diseases.
  • An individual subject can elect to have his or her own stem cell populations collected from the peripheral blood, processed and preserved, while he or she is in healthy state, for future distribution for his or her healthcare needs.
  • peripheral blood of a subject by the methods as disclosed herein are "banked” for future use, at a stem cell bank or depository or storage facility, or any place where the target stem cell populations are kept for safekeeping.
  • the storage facility may be designed in such a way that the target stem cell populations collected from the peripheral blood of a subject by the methods as disclosed herein are kept safe in the event of a catastrophic event such as a nuclear attack.
  • the storage facility might be underground, in caves or in silos. In other embodiments, it may be on the side of a mountain or in outer space.
  • the storage facility may be encased in a shielding material such as lead.
  • Step A involves administrating one or more mobilizing agents to a subject to increase the amount of stem cell populations in the peripheral blood of the subject.
  • Step B involves collecting at least one population of target stem cell populations from the peripheral blood by the methods as disclosed herein using an elutriation process, wherein said subject has no immediate perceived health condition, e.g., no condition where the subject is requiring treatment using his autologous transplantation of their own target stem cell populations.
  • Step C involves preserving the at least one population of a target stem cell population collected from the peripheral blood by the methods as disclosed herein as a preserved populations of cells.
  • Step D involves retrieving the preserved populations of target stem cells collected by the methods as disclosed herein for autologous transplantation of tire target stem cells into the subject.
  • Another aspect of the present invention relates to the use of the target stem cell
  • Such assays are currently well known in the art, and include high throughput screens where a compound, agent, or environmental stimulus is contacted with a stem cell and the effect on the function or viability of the compound, agent or environmental stimulus on the function (e.g., differentiation potential, propagation, survival) and viability can be measured, and the result compared to a reference control sample (e.g., in the absence of a compound, or a positive control sample, such as the presence of BMP6 or other growth factor and the like).
  • a reference control sample e.g., in the absence of a compound, or a positive control sample, such as the presence of BMP6 or other growth factor and the like.
  • a target stem cell population obtained without using positive selection methods from a subject's peripheral blood by the methods as disclosed herein can be used as an individualized assay for the study and understanding of signaling pathways of the subject's own stem cell populations' growth and differentiation.
  • the use of the subject's target stem cells obtained by the methods as disclosed herein are useful to aid the development of therapeutic applications for congenital and adult heart failure.
  • the use of such subject's target stem cells obtained by the methods as disclosed herein enables the study of specific differentiation into different lineages, e.g., osteogenic lineages or cardiac lineages, without the need and complexity of time consuming animal models.
  • the subject's target stem cells obtained by the methods as disclosed herein can be genetically modified to carry specific disease and/or pathological traits and phenotypes of a particular disease or disorder.
  • the assay comprises a plurality of the subject's different target stem cell populations obtained by the methods as disclosed herein, or their differentiated progeny.
  • the assay can be used for the study of differentiation pathways of a subject's different target stem cell populations, for example but not limited to the differentiation along the cardiomyocyte lineage, smooth muscle lineage, endothelial lineage, and subpopulations of these lineages.
  • the assay can be used to study a pathological characteristic of the subject's different target stem cell populations, for example, a disease and/or genetic characteristic associated with a disease or disorder.
  • the disease or disorder is a wound healing disorder, bone disorder or a cardiovascular disorder or disease.
  • the subject's different target stem cell populations have been genetically engineered to comprise the characteristics associated with a disease or disorder.
  • Such methods to genetically engineer stem cells are well known by those in the art, and include introducing nucleic acids into the cell by means of transfection by, for example, but not limited to, use of viral vectors or by other means known in the art.
  • the use of the MSCs provide a tool for pharmacogenetic analysis of the target stem cell populations.
  • a subject's target stem cells may harbor a particular variant which results in a distinct pathological characteristic.
  • the stem cells may have an undesirable pathological characteristic, e.g., mutation and/or polymorphism, which contributes to disease pathology, or results in a poor or enhanced response to a therapeutic drug.
  • the target stem cells obtained by the methods disclosed herein can be used to assess the response of the subject's stem cells to particular compounds, agents, and in some cases be useful for determining treatment regimens, (e.g., effective treatment drugs, doses and durations) and in some cases, prognosis for disease.
  • the methods of the invention can be used to screen the target stem cells for agents which alleviate the pathology, or agents which positively affect the propagation or function of the target stem cells.
  • the methods of the invention can be used to screen for agents which cause a different response on a population of the subject's target stem cells (due to an inherent genetic make up or a particular mutation and/or polymorphism) as compared with stem cells from other subjects (e.g., without the mutation and/or polymorphism), therefore the methods can be used for example, to assess an effect of a particular drug and/or agent on subject's stem cell population as compared to other people and/or stem cell populations, therefore acting as a high-throughput screen for personalized medicine and/or pharmacogenetics.
  • the manner in which the target stem cells respond to an agent, particularly a pharmacologic agent, including the timing of responses, is an important reflection of the physiologic state of the cell.
  • the subject's different target stem cell populations can be easily manipulated in experimental systems that offer the advantages of targeted lineage differentiation as well as clonal homogeneity and the ability to manipulate external environments. Furthermore, due to the ethical unacceptability of experimentally altering a human germ line, the ES cell transgenic route is not available for experiments that involve the manipulation of human genes. Gene targeting in a human subject's different target stem cell populations allows important applications in areas where the system cannot be tested in vivo due to ethical issues, and can adequately recapitulate human biology or disease processes for that particular subject or individual.
  • the subject's different target stem cell populations can be used to prepare a cDNA library relatively uncontaminated with cDNA that is preferentially expressed in cells from other lineages.
  • the subject's different target stem cell populations can also be used to prepare
  • Polyclonal antibodies that are specific for markers of cardiomyocytes and their precursors.
  • Polyclonal antibodies can be prepared by injecting a vertebrate animal with cells of this invention in an immunogenic form. Production of monoclonal antibodies is described in such standard references as U.S. Pat. Nos. 4,491,632; 4,472,500 and 4,444,887, and Methods in
  • Specific antibody molecules can also be produced by contacting a library of immunocompetent cells or viral particles with the target antigen, and growing out positively selected clones. See Marks et al, New Eng. J. Med. 335:730, 1996, and
  • the antibodies in turn can be used to identify or rescue (for example, restore the
  • phenotype cells of a desired phenotype from a mixed cell population, for purposes such as costaining during immunodiagnosis using tissue samples, and isolating precursor cells from a terminally differentiated subject's different target stem cell populations and cells of other lineages.
  • the subject's different target stem cell populations can be used for the examination of the gene expression profile during and following differentiation of the subject's different target stem cell populations.
  • the expressed set of genes may be compared against other subsets of stem cell populations from the same or a different subject or control stem cell line sample as known in the art.
  • Any suitable qualitative or quantitative methods known in the art for detecting specific mRNAs can be used.
  • mRNA can be detected by, for example, hybridization to a microarray, in situ hybridization in tissue sections, by reverse transcriptase-PCR, or in Northern blots containing poly A+ mRNA.
  • One of skill in the art can readily use these methods to determine differences in the molecular size or amount of m NA transcripts between two samples.
  • mRNA expression levels in a sample can be determined by generation of a library of expressed sequence tags (ESTs) from a sample. Enumeration of the relative representation of ESTs within the library can be used to approximate the relative representation of a gene transcript within the starting sample. The results of EST analysis of a test sample can then be compared to EST analysis of a reference sample to determine the relative expression levels of a selected polynucleotide, particularly a polynucleotide corresponding to one or more of the differentially expressed genes described herein.
  • ESTs expressed sequence tags
  • gene expression in a test sample can be performed using serial analysis of gene expression (SAGE) methodology (Velculescu et al., Science (1995) 270:484).
  • SAGE serial analysis of gene expression
  • SAGE involves the isolation of short unique sequence tags from a specific location within each transcript. The sequence tags are concatenated, cloned, and sequenced. The frequency of particular transcripts within the starting sample is reflected by the number of times the associated sequence tag is encountered with the sequence population.
  • Gene expression in a test sample can also be analyzed using differential display (DD) methodology.
  • DD differential display
  • fragments defined by specific sequence delimiters e.g., restriction enzyme sites
  • the relative representation of an expressed gene within a sample can then be estimated based on the relative
  • DD fragment-associated fragment-associated with that gene within the pool of all possible fragments.
  • Methods and compositions for carrying out DD are well known in the art, see, e.g., U.S. Patent No. 5,776,683; and U.S. Patent No. 5,807,680.
  • gene expression can be estimated in a sample using hybridization analysis, which is based on the specificity of nucleotide interactions. Oligonucleotides or cDNA can be used to selectively identify or capture DNA or RNA of specific sequence composition, and the amount of RNA or cDNA hybridized to a known capture sequence determined qualitatively or quantitatively, to provide information about the relative representation of a particular message within the pool of cellular messages in a sample.
  • Hybridization analysis can be designed to allow for concurrent screening of the relative expression of hundreds to thousands of genes by using, for example, array-based technologies having high density formats, including filters, microscope slides, or microchips, or solution-based technologies that use spectroscopic analysis (e.g., mass spectrometry).
  • array-based technologies having high density formats, including filters, microscope slides, or microchips
  • solution-based technologies that use spectroscopic analysis (e.g., mass spectrometry).
  • spectroscopic analysis e.g., mass spectrometry
  • Hybridization to arrays may be performed, where the arrays can be produced according to any suitable methods known in the art. For example, methods of producing large arrays of oligonucleotides are described in U.S. Patent No. 5,134,854, and U.S. Patent No. 5,445,934 using light-directed synthesis techniques. Using a computer-controlled system, a
  • heterogeneous array of monomers is converted, through simultaneous coupling at a number of reaction sites, into a heterogeneous array of polymers.
  • microarrays are generated by deposition of pre-synthesized oligonucleotides onto a solid substrate, for example, as described in PCT published application no. WO 95/35505.
  • Methods for collection of data from hybridization of samples with an array are also well known in the art.
  • the polynucleotides of the cell samples can be generated using a detectable fluorescent label, and hybridization of the polynucleotides in the samples detected by scanning the microarrays for the presence of the detectable label.
  • Methods and devices for detecting fluorescently marked targets on devices are known in the art.
  • such detection devices include a microscope and light source for directing light at a substrate.
  • a photon counter detects fluorescence from the substrate, while an x-y translation stage varies the location of the substrate.
  • a confocal detection device that can be used in the subject methods is described in U.S. Patent No. 5,631,734.
  • a scanning laser microscope is described in Shalon et al., Genome Res. (1996) 6:639.
  • a scan, using the appropriate excitation line, is performed for each fluorophore used.
  • the digital images generated from the scan are then combined for subsequent analysis. For any particular array element, the ratio of the fluorescent signal from one sample is compared to the fluorescent signal from another sample, and the relative signal intensity determined.
  • data analysis can include the steps of determining fluorescent intensity as a function of substrate position from the data collected, removing outliers, i.e., data deviating from a predetermined statistical distribution, and calculating the relative binding affinity of the targets from the remaining data.
  • the resulting data can be displayed as an image with the intensity in each region varying according to the binding affinity between targets and probes. Pattern matching can be performed manually or can be performed using a computer program.
  • a stem cell population or a combination of stem cell populations are contacted with the agent of interest, and the effect of the agent assessed by monitoring output parameters, such as expression of markers, cell viability, differentiation characteristics, multipotent capacity and the like.
  • the target stem cells may be freshly isolated using the methods as disclosed herein, or in some embodiments, cultured, cryopreserved, or genetically engineered prior to using in an assay.
  • the target stem cells can be environmentally induced variants of clonal cultures: e.g., split into independent cultures and grown under distinct conditions, for example, with or without virus; in the presence or absence of other cytokines or combinations thereof.
  • the target stem cells may be variants with a desired pathological characteristic.
  • the target stem cells may be from the subject (i.e., autologous) they have a desired pathological characteristic, e.g., mutation and/or polymorphism which contribute to disease pathology. Therefore, the pool of target stem cells obtained by the methods as disclosed herein can be used to assess the subject's target stem cells' response to particular compounds, agents, and in some cases may be useful for a prognosis for disease.
  • the methods of the invention can be used to screen the target stem cell populations for agents which alleviate the pathology.
  • the methods of the invention can be used to screen for agents where some target stem cell populations comprising a particular mutation and/or polymorphism respond differently as compared with the same type of stem cells without the mutation and/or polymorphism. Therefore, the methods can be used, for example, to assess an effect of a particular drug and/or agent on a subject's target stem cell population as compared to other people and/or cells, therefore acting as a high-throughput screen for personalized medicine and/or pharmacogenetics.
  • the manner in which cells respond to an agent, particularly a pharmacologic agent, including the timing of responses, is an important reflection of the physiologic state of the cell.
  • the agent used in the screening method can be selected from a group of a chemical, small molecule, chemical entity, nucleic acid sequences, an action, nucleic acid analogues or protein or polypeptide or analogue of fragment thereof.
  • the nucleic acid is DNA or RNA, and nucleic acid analogues, for example, can be PNA, pcPNA and LNA.
  • a nucleic acid may be single or double stranded, and can be selected from a group comprising; nucleic acid encoding a protein of interest, oligonucleotides, PNA, etc.
  • nucleic acid sequences include, for example, but are not limited to, nucleic acid sequence encoding proteins that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory' nucleic acid sequences, for example, but not limited to RNAi, shR Ai, siRNA, micro RNAi (mRNAi), antisense oligonucleotides, etc..
  • a protein and/or peptide agent or fragment thereof can be any protein of interest, for example, but not limited to, mutated proteins, therapeutic proteins, or truncated proteins, wherein the protein is normally absent or expressed at lower levels in the cell.
  • Proteins of interest can be selected from a group comprising: mutated proteins, genetically engineered proteins, peptides, synthetic peptides, recombinant proteins, chimeric proteins, antibodies, humanized proteins, humanized antibodies, chimeric antibodies, modified proteins and fragments thereof.
  • the agent may be applied to the media, where it contacts the cell (such as stem cells and/or mesenchymal cells) and induces its effects.
  • the agent may be intracellular, i.e., within the cell (such as stem cell and/or mesenchymal cells), as a result of introduction of the nucleic acid sequence into the cell and its transcription, resulting in the production of the nucleic acid and/or protein agent within the cell.
  • an agent also encompasses any action and/or event the cells are subjected to.
  • an action can comprise any action that triggers a physiological change in the cell, for example, but not limited to, heat-shock, ionizing irradiation, cold-shock, electrical impulse, light and/or wavelength exposure, UV exposure, pressure, stretching action, increased and/or decreased oxygen exposure, exposure to reactive oxygen species (ROS), ischemic conditions, fluorescence exposure etc.
  • ROS reactive oxygen species
  • Environmental stimuli also include intrinsic environmental stimuli defined below.
  • the exposure to agent may be continuous or non-continuous.
  • agent refers to any chemical, entity or moiety, including without limitation synthetic and naturally-occurring non-proteinaceous entities.
  • the compound of interest is a small molecule having a chemical moiety.
  • chemical moieties included unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties including macrolides, leptomycins and related natural products or analogues thereof.
  • Compounds can be known to have a desired activity and/or property, or can be selected from a library of diverse compounds.
  • the agent is an agent of interest including known and unknown compounds that encompass numerous chemical classes, primarily organic molecules, which may include organometallic molecules, inorganic molecules, genetic sequences, etc..
  • An important aspect of the invention is to evaluate candidate drugs, including toxicity testing and the like.
  • Candidate agents also include organic molecules comprising functional groups necessary for structural interactions, particularly hydrogen bonding, and typically include at least an amine, carbonyi, hydroxyl or carboxyl group, frequently at least two of the functional chemical groups.
  • the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate agents are also found among biomolecules, including peptides, polynucleotides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • agents are pharmacologically active drugs, genetically active agents, and others.
  • Compounds of interest include, for example, chemotherapeutic agents, hormones or hormone antagonists, growth factors or recombinant growth factors and fragments and variants thereof.
  • chemotherapeutic agents include, for example, chemotherapeutic agents, hormones or hormone antagonists, growth factors or recombinant growth factors and fragments and variants thereof.
  • exemplary of pharmaceutical agents suitable for this invention are those described in, "The Pharmacological Basis of Therapeutics," Goodman and Gilman, McGraw-Hill, New York, N.Y., (1 96), Ninth edition, under the sections:
  • the agents include all of the classes of molecules described above, and may further comprise samples of unknown content. Of interest are complex mixtures of naturally occurring compounds derived from natural sources such as plants. While many samples will comprise compounds in solution, solid samples that can be dissolved in a suitable solvent may also be assayed. Samples of interest include environmental samples, e.g., ground water, sea water, mining waste, etc.; biological samples, e.g., lysates prepared from crops, tissue samples, etc.; manufacturing samples, e.g., time course during preparation of
  • Samples of interest include compounds being assessed for potential therapeutic value, i.e., drug candidates.
  • Parameters are quantifiable components of target stem cells, particularly components that can be accurately measured, desirably in a high throughput system.
  • a parameter can be any cell component or cell product including cell surface determinant, receptor, protein or conformational or posttranslational modification thereof, lipid, carbohydrate, organic or inorganic molecule, nucleic acid, e.g., mRNA, DNA, etc. or a portion derived from such a cell component or combinations thereof. While most parameters will provide a quantitative readout, in some instances a semi-quantitative or qualitative result will be acceptable.
  • Readouts may include a single determined value, or may include mean, median value or the variance, etc.. Characteristically a range of parameter readout values will be obtained for each parameter from a multiplicity of the same assays. Variability is expected and a range of values for each of the set of test parameters will be obtained using standard statistical methods with a common statistical method used to provide single values.
  • Compounds, including candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds, including biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc., to produce structural analogs.
  • Agents are screened for effect on the stem cell population by adding the agent to at least one and usually a plurality of stem cell samples, usually in conjunction with cells lacking the agent.
  • the change in parameters in response to the agent is measured, and the result evaluated by comparison to reference cultures, e.g., in the presence and absence of the agent, obtained with other agents, etc..
  • the agents are conveniently added in solution, or readily soluble form, to the medium of stem cells in culture.
  • the agents may be added in a flow-through system, as a stream, intermittent or continuous, or alternatively, by adding a bolus of the compound, singly or incrementally, to an otherwise static solution.
  • a flow-through system two fluids are used, where one is a physiologically neutral solution, and the other is the same solution with the test compound added. The first fluid is passed over the stem cells, followed by the second.
  • a bolus of the test compound is added to the volume of medium surrounding the cells.
  • agent formulations do not include additional components, such as preservatives, that may have a significant effect on the overall formulation.
  • preferred formulations consist essentially of a biologically active compound and a physiologically acceptable carrier, e.g., water, ethanol, DMSO, etc..
  • a physiologically acceptable carrier e.g., water, ethanol, DMSO, etc.
  • the formulation may consist essentially of the compound itself.
  • a plurality of assays may be run in parallel with different agent concentrations to obtain a differential response to the various concentrations.
  • determining the effective concentration of an agent typically uses a range of concentrations resulting from 1 :10, or other log scale, dilutions.
  • the concentrations may be further refined with a second series of dilutions, if necessary.
  • one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection of the agent or at or below the concentration of agent that does not give a detectable change in the phenotype.
  • the stem cell population used in the screen can be manipulated to express desired gene products.
  • Gene therapy can be used to either modify a cell to replace a gene product or add or knockdown a gene product.
  • the genetic engineering is done to facilitate regeneration of tissue, to treat disease, or to improve survival of the cells following implantation into a subject (e.g., prevent rejection).
  • the mesenchymal cells are genetically engineered and transfected prior to their use as a feeder layer for the stem cells, or alternatively, the mesenchymal cells can be transfected while they function as feeder layer for stem cells. Techniques for transfecting cells are known in the art.
  • genes which would convey beneficial properties to the transfected mesenchymal cells or, more indirectly, to the recipient stem cells and/or subject if the stem cells are used in transplantation (discussed in more detail below).
  • the added gene may ultimately remain in the recipient cell and all its progeny, or may only remain transiently, depending on the embodiment.
  • genes encoding angiogenic factors could be transfected into progenitor cells isolated from smooth muscle. Such genes would be useful for inducing collateral blood vessel formation as the smooth muscle tissue is regenerated. It some situations, it may be desirable to transfect the cell with more than one gene.
  • the gene product preferably contains a secretory signal sequence that facilitates secretion of the protein.
  • a skilled artisan could either select an angiogenic protein with a native signal sequence, e.g., VEGF, or can modify the gene product to contain such a sequence using routine genetic manipulation (see, e.g., Nabel et al, 1993).
  • the desired gene can be transfected into the cell using a variety of techniques.
  • the gene is transfected into the cell using an expression vector.
  • Suitable expression vectors include plasmid vectors (such as those available from Stratagene, Madison Wis.), viral vectors (such as replication defective retroviral vectors, herpes virus, adenovirus, adenovirus associated virus, and lentivirus), and non-viral vectors (such as liposomes or receptor ligands).
  • the present invention includes an ELUTRA® centrifuge manufactured by Gambro BCT, Inc. of
  • PB cells were subjected to elutriation to separate/enrich MSCs.
  • a group of 28 donors were studied after mobilization for two consecutive days of G-CSF injection (48(fyg/day subcutaneously) and collection of total nucleated cells (TNC) by apheresis for 3-4 hours on the third day.
  • CFU-F Assay ELUTRA® fractions were tested for a possible enrichment in MSCs by plating cells in a CFU-F assay. 2 million cells per each fraction were plated onto a 100-mm tissue culture dish and supplemented with 10 mL of DMEM + 10% FBS media. Cultures were left undisturbed in a 37° C incubator with 5% CO 2 for two weeks. After two weeks, cultures were fixed with methanol and stained with Giemsa. Adherent colonies characteristic of MSCs developed from fractions 4 and 5 ( Figure 4).
  • Staining of fractionated apheresis populations Flow cytometry analysis was used to determine whether certain ELUTRA fractions were enriched in MSCs. Samples from the ELUTRA® fractions were stained using two panels of antibodies: panel 1: CD45, CD105, CD90, and panel 2: CD271. Staining was performed in PBS containing 0.5% Human Serum Albumin for 30 minutes on ice. Cells were washed and analyzed by FACS as CD457CD105 ⁇ , CD457CD90 " and CD457CD27 . Fractions 4 and 5 were enriched for cells characterized by CD457CD105 + , CD457CD90 + , and CD271 + as compared to the aphesis product starting population ( Figure 5).
  • G-CSF mobilized MSCs into peripheral blood.
  • Figure 6 shows that the proportion of MSCs increased post mobilization and in the resulting apheresis product.
  • MSCs were identified by detecting cells expressing CD457CD317CD105 + /CD73 + or CD457CD34 " /CD907CD105 + /CD29 + .

Abstract

La présente invention concerne un procédé pour isoler au cours d'au moins une étape plusieurs populations de cellules souches à partir d'un échantillon de sang périphérique sans recourir à la sélection positive, ce qui permet ainsi de mettre en œuvre un procédé "sans contact" pour isoler différentes populations de cellules souches sans contaminer les populations de cellules souches avec des agents de sélection positive (par exemple, des anticorps, etc.). La présente invention concerne notamment une séparation suivant le débit de populations de cellules souches en procédant à une élutriation (séparation basée sur la taille des cellules) pour exclure négativement les cellules en fonction de leur taille. Différentes fractions ou sous-fractions comprennent une augmentation du rendement des très petites cellules souches de type embryonnaire (VSEL), des MSC ou des HSC comparé à un produit d'aphérèse non fractionné.
PCT/US2010/058974 2009-12-04 2010-12-03 Procédé pour isoler des populations de cellules souches du sang périphérique en procédant à une séparation basée sur leur taille (élutriation) WO2011069117A1 (fr)

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013096778A1 (fr) * 2011-12-21 2013-06-27 Lonza Walkersville Inc. Procédé évolutif de concentration cellulaire thérapeutique et de clairance résiduelle
EP2773746A4 (fr) * 2011-11-01 2015-08-05 Neostem Inc Compositions de cellules souches mésenchymateuses adultes (msc) et procédés de préparation associés
WO2017153974A1 (fr) * 2016-03-07 2017-09-14 Caladrius Biosciences, Inc. Système fermé pour le marquage et la sélection de cellules vivantes
US20180179490A1 (en) * 2016-12-27 2018-06-28 Miltenyi Biotec Gmbh CELL COMPOSITION DEPLETED FROM TCRab and CD45RA POSITIVE CELLS
WO2019081765A1 (fr) * 2017-10-26 2019-05-02 Marti Guy Procédé et appareil de purification de cellules souches mésenchymateuses
WO2020214400A1 (fr) * 2019-04-15 2020-10-22 Ossium Health, Inc. Système et procédé d'extraction et de cryopréservation de moelle osseuse
US11072777B2 (en) 2016-03-04 2021-07-27 University Of Louisville Research Foundation, Inc. Methods and compositions for ex vivo expansion of very small embryonic-like stem cells (VSELs)
US20210395693A1 (en) * 2018-10-26 2021-12-23 Guy MARTI Method and apparatus for mesenchymal stem cells isolation and purification
US11312940B2 (en) 2015-08-31 2022-04-26 University Of Louisville Research Foundation, Inc. Progenitor cells and methods for preparing and using the same
US11744243B2 (en) 2020-10-14 2023-09-05 Ossium Health, Inc. Systems and methods for extraction and cryopreservation of bone marrow
US11786558B2 (en) 2020-12-18 2023-10-17 Ossium Health, Inc. Methods of cell therapies
US11896005B2 (en) 2020-07-18 2024-02-13 Ossium Health, Inc. Warming cryopreserved bone

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050250204A1 (en) * 2001-12-05 2005-11-10 Gambro, Inc. Methods and apparatus for separation of particles
US20070154877A1 (en) * 2005-12-07 2007-07-05 Gerold Schuler Method for the direct culture of dendritic cells without a preceding centrifugation step
US20090220466A1 (en) * 2005-12-08 2009-09-03 Mariusz Ratajczak Very small embryonic-like (vsel) stem cells and methods of isolating and using the same
US20100095669A1 (en) * 2007-03-16 2010-04-22 Csir Wax Actuator and a Method of Actuating by Means of a Wax Actuator

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050250204A1 (en) * 2001-12-05 2005-11-10 Gambro, Inc. Methods and apparatus for separation of particles
US20070154877A1 (en) * 2005-12-07 2007-07-05 Gerold Schuler Method for the direct culture of dendritic cells without a preceding centrifugation step
US20090220466A1 (en) * 2005-12-08 2009-09-03 Mariusz Ratajczak Very small embryonic-like (vsel) stem cells and methods of isolating and using the same
US20100095669A1 (en) * 2007-03-16 2010-04-22 Csir Wax Actuator and a Method of Actuating by Means of a Wax Actuator

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2773746A4 (fr) * 2011-11-01 2015-08-05 Neostem Inc Compositions de cellules souches mésenchymateuses adultes (msc) et procédés de préparation associés
WO2013096778A1 (fr) * 2011-12-21 2013-06-27 Lonza Walkersville Inc. Procédé évolutif de concentration cellulaire thérapeutique et de clairance résiduelle
US10385307B2 (en) 2011-12-21 2019-08-20 Lonza Walkersville, Inc. Scalable process for therapeutic cell concentration and residual clearance
US11312940B2 (en) 2015-08-31 2022-04-26 University Of Louisville Research Foundation, Inc. Progenitor cells and methods for preparing and using the same
US11072777B2 (en) 2016-03-04 2021-07-27 University Of Louisville Research Foundation, Inc. Methods and compositions for ex vivo expansion of very small embryonic-like stem cells (VSELs)
WO2017153974A1 (fr) * 2016-03-07 2017-09-14 Caladrius Biosciences, Inc. Système fermé pour le marquage et la sélection de cellules vivantes
GB2565664A (en) * 2016-03-07 2019-02-20 Hitachi Chemical Advanced Therapeutics Solutions Llc A closed system for labelling and selecting live cells
US20180179490A1 (en) * 2016-12-27 2018-06-28 Miltenyi Biotec Gmbh CELL COMPOSITION DEPLETED FROM TCRab and CD45RA POSITIVE CELLS
US11618887B2 (en) 2017-10-26 2023-04-04 Guy MARTI Method and apparatus for mesenchymal stem cells purification
WO2019081765A1 (fr) * 2017-10-26 2019-05-02 Marti Guy Procédé et appareil de purification de cellules souches mésenchymateuses
US20210395693A1 (en) * 2018-10-26 2021-12-23 Guy MARTI Method and apparatus for mesenchymal stem cells isolation and purification
US10995318B2 (en) 2019-04-15 2021-05-04 Ossium Health, Inc. System and method for extraction and cryopreservation of bone marrow
US11085024B2 (en) 2019-04-15 2021-08-10 Ossium Health, Inc. System and method for extraction and cryopreservation of bone marrow
US11104882B2 (en) 2019-04-15 2021-08-31 Ossium Health, Inc. System and method for extraction and cryopreservation of bone marrow
WO2020214400A1 (fr) * 2019-04-15 2020-10-22 Ossium Health, Inc. Système et procédé d'extraction et de cryopréservation de moelle osseuse
US11447750B2 (en) 2019-04-15 2022-09-20 Ossium Health, Inc. System and method for extraction and cryopreservation of bone marrow
US11697799B2 (en) 2019-04-15 2023-07-11 Ossium Health, Inc. System and method for extraction and cryopreservation of bone marrow
US11702637B2 (en) 2019-04-15 2023-07-18 Ossium Health, Inc. System and method for extraction and cryopreservation of bone marrow
US11896005B2 (en) 2020-07-18 2024-02-13 Ossium Health, Inc. Warming cryopreserved bone
US11744243B2 (en) 2020-10-14 2023-09-05 Ossium Health, Inc. Systems and methods for extraction and cryopreservation of bone marrow
US11786558B2 (en) 2020-12-18 2023-10-17 Ossium Health, Inc. Methods of cell therapies

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