TW201130978A - Method of isolation of stem cell populations from peripheral blood using sized-based separation (elutriation) - Google Patents

Abstract

The present invention relates to a method for isolating multiple populations of stem cells in at least one step from a single peripheral blood sample or mobilized peripheral blood sample without using positive 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.). In particular, the present invention relates to 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 cells of interest. In some embodiments, different fractions or subfractions comprise an increase in yield of Very Small Embryonic-like Stem cells (VSELs), MSCs or HSCs as compared to non-fractionated apheresis product. The isolated 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. 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, or lifestyle on the function and viability of different stem cell populations, either alone or as a combination of different stem cells.

Description

201130978 VI. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The subject matter of the present invention relates generally to cell separation using a device specific for cell size and density. The biological sample from which the cells are derived 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 provides an advantageous method of separating peripheral blood into a subset of desired stem cell populations. [Prior Art] Adult stem cells are undifferentiated cells found in differentiated tissues. It has the ability to self-renew and become specialized to produce all cell types of the tissue from which it originates and, in an appropriate environment, also become specialized cells of different tissues. Adult stem cells are capable of self-renewal in one of the organisms. The source of adult stem cells has been found in bone marrow, blood flow, cornea of the eye and retina, pulp, liver, skin, gastrointestinal tract, adipose tissue and pancreas. Stem cells provide the possibility to replenish renewable sources of cells and tissues to treat countless diseases, conditions and disability, especially in organisms with low cell turnover,

The injury produces a rapid and strong reaction.

The phenomenon of plasticity. Recently, it has been considered that it is derived from bone marrow or peripheral blood (packaging cannot produce cells of different tissue types. A large number of clinical studies have been confirmed in recent years. Plasticity is an adult stem cell from a specific cell 152717.doc 201130978 strained into other cell types Capability. In other words, stem cells from bone marrow or self-circulating blood do have the ability to differentiate into other cell types, such as heart cells or nerve cells. Because this can be (4) 'so human adult stem cells are derived from other donors or embryonic stem cells. The perfect ethical and ethical alternative to stem cells. Adult stem cell transplants (bone marrow grafts) have been successfully used to treat cancer (such as leukemia multiple myeloma and lymphoma) for more than 40 years, and these grafts are now open for regeneration. The door to restorative therapeutics. 6 people who have been diagnosed with a disease requiring bone marrow transplantation cannot find a healthy partner and have a very low chance of finding a suitable donor. Therefore, it is advisable to wait until the bone marrow transplant is needed. Individuals collect stem cells, which can store δ metaphyseal stem cells so that they are not Autologous transplantation is performed at a time. Therefore, it is appropriate to collect stem cells from healthy individuals when stem cells may not have been damaged or planned due to disease. In addition, stem cells may be collected from any healthy adult for future autologous transplantation for the disease or As a regenerative therapy, as well as collecting stem cells from adults with a positive family history of heart disease, diabetes, cancer, neurological disease or autoimmune disorders for use in therapeutic use. Autologous stem cell transplantation (receiving self-stem cells) There are significant clinical and economic advantages' including non-immune rejection issues, including, for example, the production of host-versus-versus-infected disease (HVGD) (the recipient's immune system treats donor cells as "non-self" and destroys transplanted cells) or graft resistance Host disease (GVHD) (donor cells reject "non-self" recipient cells and destroy host (recipient) cells.) In addition, transplantation of individuals with their own stem cells (autologous) is accepted by 152717.doc 201130978 Other people's stem cells (heterologous) are much faster, safer and less expensive The ability to identify, characterize, and purify stem cell subpopulations is critical in autologous transplantation therapies and in a variety of biological and medical applications, often forming the starting point for research protocols and the basis of current and emerging clinical protocols. There are numerous applications in areas such as medicine, biotechnology, biomedical research, environmental monitoring, and biological/chemical warfare defense. For example, cell separation can lead to viable life-saving procedures (such as autologous bone marrow transplantation to remedy advanced cancer). Carcinogenic metastatic cells must be removed from the patient (4) (Fischer, 丨 993). Highly purified cell subsets in other applications, such as signaling studies between blood cells (Stout, 1993; Cantrell et al., (10) 2) Studies that are otherwise impossible are allowed. In general, current methods of cell sorting most often utilize specific immunological targets (Smeland et al., 1992) or receptor-ligand interactions (〇11 to 3 et al., 1976). Differences in cell density or in some cases (B〇yum, 1974) to isolate specific cells or cell populations. However, the prior art often does not and therefore requires a sorting device capable of identifying and selectively manipulating stem cells via novel physical properties. In addition, the technique of isolating stem cell populations from humans typically separates only one stem cell population while abandoning other stem cell populations, and often uses positive selection techniques that result in contamination of the collected stem cell population by the agents used in the forward selection method. The application of AC electrodynamic principles has been used for electroporation (R〇T) methods (Arn〇ld and Zimmermann, 1982; Fuhr, 1985; H61zel and Lamprecht, 1992; Wang et al., 1994) and for cell identification and sorting ( Hagedorn I52717.doc 201130978 Specialist '1992, Huang special person' 1993; Gascoyne et al, 1992; Gascoyne et al, 1994; Huang et al, 1992) to dielectrically characterize mammalian cells. In these techniques, cells become electrically polarized when subjected to an AC electric field. In the ROT, a rotating electric field is applied and the interaction between the cell polarization and the applied field causes the cells to rotate. If the field is not uniform, the cells are subjected to lateral dielectrophoresis (DEP) forces whose frequency response varies with their inherent electrical properties (Gascoyne et al., 1992). Moreover, these characteristics strongly depend on the composition and organization of the cells, reflecting the characteristics of the cell morphology and phenotype. Cells with different polarizations can thus undergo differentiating forces in a non-uniform electric field (Becker et al., 1994; Becker et al., 1995). Dielectrophoretic motion analysis of mammalian cells as a function of frequency of administration allows detection of cell membrane biophysics. Learn parameters such as capacitance and surface conduction. Because DEP forces effectively map biophysical properties to directions and magnitudes reflect the translational forces of cellular properties, DEp forces can induce separation between particles of different characteristics. For example, DEP has been used on a microscopic scale to isolate bacteria from red blood cells (Markx et al., 1994), to isolate survivors from non-live yeast cells (Wang et al., 1993) and to isolate red leukemia cells from red blood cells (Huang et al., 1992). However, the difference in electrode type of cell types in their various mixtures is greater than would be expected in many typical cell sorting applications. Field flow fractionation (FFF) has also been used to separate materials using particle density, size, volume, diffusivity, and surface charge as parameters (Giddings, 1993). This technique can be used to separate many different types of materials ranging in size from about 1 nanometer to greater than about 1 micron, which can include, for example, biological and non-biological materials. Separation by field flow fractionation is performed by 152717.doc 201130978 by the differential interception in the flow through the thin channel to generate e FFF technology combined chromatography, electrophoresis and ultracentrifugation elements, and its use when the fluid flows through The flow rate pattern established in the thin channel during the chamber. This velocity profile can be, for example, linear or parabolic. A field is then applied at right angles to the flow and is used to push the material into different displacements within the flow pattern. Substances that are displaced at different locations within the velocity profile are carried by fluid flowing through the chamber at different rates. Fields can be based on settling, crossover, temperature gradients, centrifugal forces, and the like. However, these techniques have the disadvantage that the resulting population of cells is not sufficiently pure 'too slow, or too limited to the spectrum of target cells or other substances. In the medical field, stem cell populations can be enriched from peripheral blood. Whole blood is composed of various liquid components and particle components. The liquid part of the liquid is mainly composed of plasma, and the particle components include red blood cells (red bl00d ceu; erythrocyte; RBC), white blood cells (White bl〇〇d cell; leuk〇cyte; boundary 8 and platelets (1 ^ ^); More than 1_〇„11)〇(1)^) and stem cells such as, but not limited to, hematopoietic stem cells (HSC), mesenchymal stem cells (MSC), and other small stem cells, such as very small embryo-like (VSEL) stem cells. The components have a similar density' but the average density relationship in descending order of density is as follows: red blood cells, white blood cells, platelets, and plasma. In addition, the particle components are in descending order of magnitude as follows: white blood cells, red blood cells, and platelets. Purification devices rely on differences in density and size or surface chemistry to separate and/or transition blood components. Therefore, there is a need in the art for an easy and fast population of peripheral blood-enriched stem cells that are readily available from clinical settings. And effective methods. [Summary] 152717.doc -9· 201130978 Peripheral blood and mobilized peripheral blood contain many different types of cells, including Stem cells, such as, but not limited to, very small embryonic stem cells (VSELs), mesenchymal stem cells (MSCs), and hematopoietic stem cells (HSCs). Conventional methods for isolating stem cell populations from peripheral blood use positive and negative selection methods, for example, Cell surface markers are used in methods such as fluorescence activated cell sorting (FACS) and other immunoselective methods to select or exclude cell populations. In addition, such methods also typically separate only one stem cell population from peripheral blood samples. In one embodiment, the present invention is directed to a method of isolating a plurality of stem cell populations from a single peripheral blood sample or a mobilized peripheral blood sample in at least one step without using positive or negative selection, thus A "non-contact" method of separating different stem cell populations without contaminating the stem cell population with a positive selection agent (eg, an antibody, etc.). In particular, in one embodiment, the present invention relates to the use of a panning method (larger, mainly separation method) to exclude cells by size to separate peripheral blood into various panning knives (each containing different A stem cell population flow rate separation method for related stem cells. The inventors have demonstrated that different fractions or sub-portions contain an increase in the yield of very small embryonic stem cells (VSEL), MSC or HSC compared to the unfractionated fractionation product. In certain embodiments, the isolated population of stem cells obtained by the methods disclosed herein provides a pool of different stem cell types of a particular individual that can be maintained in culture and/or cryopreserved for future use, For example, for individual use, or for selective recombination (eg, conventional mixing) for use in individualized autologous therapeutic applications in regenerative therapies. In addition, in some embodiments, the population of stem cells that are separated from each other also provides a pool of different stem cell populations, either alone or in combination with different stem cells, for use in personalized cells based on 152717.doc 201130978, for such assays. To assess the effects of human consumption, pharmacogenetics, neurochemicals, and lifestyle on the function and viability of different stem cell populations. Thus, the present invention generally relates to methods and compositions for separating or fractionating peripheral blood into a plurality of portions. The plurality of portions comprise different stem cell populations from a single peripheral blood sample of the individual in a single collection method. The present invention relates generally to a rapid, easy, efficient, and inexpensive method of isolating stem cell populations from peripheral blood, such as peripheral blood obtained from a human subject, without the use of positive selection techniques. In certain embodiments, the peripheral blood is mobilized peripheral blood, and in some embodiments the peripheral blood line is pretreated by a stripping procedure to produce a separation product. In particular, the present invention relates to a method of isolating a plurality of different stem cell populations from a peripheral blood sample without the need to add a positive selection agent to the peripheral blood sample. Accordingly, the inventors have discovered a method of isolating a plurality of different stem cell populations from a single sample without contaminating the collected population of stem cells with a positive selection agent. In certain embodiments, the invention provides a method of fractionating peripheral blood into a plurality of portions, wherein each portion comprises a different population of target stem cells. Accordingly, the present invention is directed to a method of enriching a target stem cell population obtained from peripheral blood without using a positive selection technique. In particular, the present invention relates to a method of separating peripheral blood samples into a plurality of different portions to enable enrichment of a plurality of different target stem cell populations in different portions. In certain embodiments, the resulting portion comprising the target stem cell population can be further separated, if necessary, into a subset or subpopulation of the desired target stem cells. In some embodiments, the collected fraction comprising the target stem cell population can be subjected to a forward selection method to enrich the desired stem cell population. In cell-based therapies, it is often necessary to purify and concentrate a subset of stem cells in a sample, such as a peripheral blood sample. In some stem cell therapies, stem cells derived from bone marrow (BM) and/or peripheral blood (pB) sources are required to be collected from individuals for future use in autologous cell replacement or autologous cell based regenerative therapies. Peripheral blood, including mobilized peripheral blood, contains a number 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, minimal embryonic-like (VSEL) stem cells. Typical known methods for isolating such stem cell populations (such as Hsc, MSC, and VSEL) from peripheral blood samples include forward selection. Such selection methods include methods based on cell surface labeling, such as immunoselection, including isolation and isolation of the antibody from the surface of the target stem cell based on affinity molecules (such as antibodies or cell surface ligands). The target stem cells are isolated. Such positive separation methods are generally known in the art and include, without limitation, labor cell sorting (FACS), immunodensity separation, immunomagnetic separation, and the like. However, such conventional methods of selecting different stem cell populations either positively or negatively from a peripheral i-liquid sample cause a selection agent such as an antibody to contaminate the peripheral blood sample and the isolated stem cell population. In addition, these selection methods are also expensive, time-consuming and slow procedures, which often require skilled professionals to specialize in training and/or specialization of the technicians, as well as a separate selection method for separating stem cell populations from peripheral blood samples. Peripheral blood samples are only enriched - a target stem cell population and discarded

152717.doc •12- 201130978 He may be used for unselected or unenriched stem cell populations that are also present in peripheral blood samples. Accordingly, the present invention relates to a method for isolating or enriching a plurality of stem cell populations from peripheral blood samples without using the positive selection technique disclosed in the text, which has a comparison with the existing method of enriching stem cells from peripheral blood. Numerous advantages 'including, but not limited to, (1) enriching multiple different stem cell populations from a single peripheral blood sample, allowing collection of multiple different stem cell populations and agitation in peripheral blood to have a valuable or suitable stem cell population Feasible, (11) to thicken the stem cell population in the peripheral blood without adding other agents, thus avoiding the agent (for example, if the Shaw rich stem cell population is subsequently used for human autologous or allogenic transp丨antation, It may not meet the FDA-approved agent to contaminate the collected stem cell population or peripheral blood samples, (111) provides a method for enriching multiple different stem cell populations from peripheral blood, which can be used with little or no training. Easy, inexpensive and easy to use, without the need for special equipment, reagents or highly trained personnel To proceed in multiple places. In certain embodiments, the invention provides for the contamination of an antibody or other agent used in a method that is not commonly used for positive selection or isolation of stem cell populations, such as in an immunologically selective method, or without the need to separate and select based on cell surface markers. Cells (such as those performed in conventional stem cell isolation methods, where conventional stem cell isolation methods use immunoselection, such as fluorescence cell sorting (FACS) or magnetic bead separation after interaction of target cells with label (4)) A method for a plurality of different stem cell populations (IV) using antibodies labeled magnetic beads, biodegradable beads, non-biodegradable beads 152717.doc 201130978 and panning on the surface (including petri dishes) The affinity method of the antibody and a combination of these methods. Accordingly, the present invention relates generally to peripheral blood fractionation comprising separating cells present in a peripheral blood (PB) sample into different cell knives, wherein each portion is a heterogeneous cell population, some of which contain at least one different Target stem cell population. In certain embodiments, each PB portion comprising a heterologous cell population can be further fractionated into, for example, a population of different homologous target stem cells. For example, a PB portion comprising a heterogeneous population of cells comprising at least two populations of target stem cells (eg, HSCs and MSCs) can be further fractionated into substantially pure HSC populations, substantially pure MSC populations, and neither HSC nor A heterogeneous population of cells other than MSC. In another example, a PB portion comprising a population of heterologous cells comprising a population of target stem cells (e. g., VSEL stem cells) can be further fractionated into a substantially pure VSEL population and a heterogeneous non-VSEL cell population. Accordingly, the present invention relates to the isolation of a plurality of target stem cells from a single peripheral blood sample without the use of a positive selection technique and thus avoiding the selection or concentration of a target stem cell population necessary for contamination of the target stem cell population or peripheral blood sample. The method of the group. In some embodiments, peripheral blood fractionation is achieved by a panning method wherein the cell of the peripheral blood is determined by the flow rate of the panning device to be collected in a particular PB portion. In certain embodiments, the separation product from peripheral blood is treated at different flow rates through an ELUTRA® device or other suitable panning device or machine. In some embodiments, the flow rate used in the panning process is maintained and the centripetal acceleration force (i.e., "g") is varied to fractionate the surrounding or separate blood. 152717.doc •14- 201130978 The inventors demonstrated that the VSEL stem cell population can be enriched from peripheral blood using the methods disclosed herein, with an unfractionated isolated peripheral blood sample containing approximately 0.01% VSEL at 50 mL/min. The VSEL was enriched at flow rate to produce a partially isolated PB sample containing about at least 0.3% VSEL stem cells, demonstrating that the VSEL population was at least 30-fold more concentrated from peripheral blood. Thus, in certain embodiments, as disclosed in the Examples herein, the PB portion collected from a flow rate of 50 ml/min comprises a population of target stem cells comprising VSEL stem cells. In certain embodiments, the percentage of VSEL in the PB portion obtained from a flow rate of 50 ml/min 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%. . In certain embodiments, the VSEL concentration of the PB fraction collected from a flow rate of 50 ml/min is about 15 times, or about 20 times, or about 30 times, or about, compared to the unfractionated peripheral blood. 40 times, or about 50 times or more than about 50 times. In certain embodiments, as disclosed in the Examples herein, the PB portion collected from a flow rate greater than or equal to about 90 ml/min comprises a target stem cell population comprising HSCs and MSCs. In certain embodiments, the percentage of HSC or MSC in the PB portion obtained from a flow rate greater than or equal to about 90 ml/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 more than about 50%. In one aspect, the invention provides a method of isolating peripheral blood to obtain at least four target cell populations without using a positive selection technique, comprising: (a) flowing peripheral blood samples from the individual through the panning a fluid chamber of the washing device, wherein a first flow rate is selected, wherein cells in the peripheral blood that are larger than the smallest cells will flow through, and smaller cells are trapped in the fluid chamber 152717.doc 15 201130978; (b) in the absence of the retained cells Carrying out the positive selection step from step (a) collecting the trapped small cells in the fluid chamber to obtain the first target cell population; (c) recycling the peripheral blood sample containing the unretained cells through the panning device Fluid chamber' and increasing the flow rate of the remaining peripheral blood sample, wherein cells in the peripheral blood that are larger than the smallest cells will flow through, and smaller cells are trapped in the fluid chamber; (d) without positive selection steps on the retained cells Collecting the trapped small cells from step (c) in the fluid chamber to obtain a second target cell population; and (e) repeating steps (c) through (d) until All cells in the peripheral blood sample are collected into the desired cell population. In certain embodiments, the first flow rate is 20 milliliters per minute or less and the first target population of cells comprises platelets. In certain embodiments, the increased flow rate is 50 ml/min or less and the target cell population comprises a minimal embryonic-like (VSEL) stem cell population. In some embodiments, the increased flow rate is 70 ml/min or 70 ml/min and the target cell population comprises a red blood cell (RBC) population. In certain embodiments, the increased flow rate is 90 ml/min or less and the target population of cells comprises a white blood cell (WBC) population. In certain embodiments, the increased flow rate is 105 ml/min or less, and the target population of cells comprises a population of hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). In certain embodiments, the methods disclosed herein do not use positive selection techniques such as immunoselection or immunological density selection, such as FACS. In certain embodiments, the methods disclosed herein do not employ a forward selection technique that utilizes antibodies or antibody fragments. In certain embodiments, a positive selection technique can be performed on a population of retentate cells (e.g., a portion of the PB comprising a population of target stem cells) to enrich the desired target stem cell population in the population of the retentate cell population of 152717.doc • 5 201130978. In a second embodiment, the method of isolating peripheral blood to obtain at least four target cell populations disclosed herein uses peripheral blood samples, such as human peripheral blood samples' including mobilized peripheral blood samples. In certain embodiments, the peripheral blood sample is obtained from a human subject after administration of the mobilizer, such as wherein the mobilizer has been administered to the human subject for at least 2 days, or 4 days or less. In certain embodiments, the peripheral blood sample is a separation product. One aspect of the present invention relates to a method of separating peripheral blood to obtain at least four target cell populations without using a positive selection technique, comprising: (a) allowing peripheral blood samples from the individual to flow through the panning Washing the fluid chamber of the device to, wherein the first flow rate is selected, wherein cells in the peripheral blood that are larger than the smallest cells will flow through, and smaller cells are trapped in the fluid chamber; without performing a positive selection step on the retained cells Collecting the occluded cells from the fluid chamber to the step (a) to obtain the first target cell population; (magicizing the peripheral blood sample containing the unretained cells to be recirculated through the fluid chamber of the panning device, and increasing the remaining periphery The flow rate of the blood sample, wherein cells in the peripheral blood that are larger than the smallest cells will flow through 'and smaller cells are trapped in the fluid chamber; (d) in the fluid chamber without performing a positive selection step on the retained cells Collecting the trapped small cells from step (c) to obtain a second target cell population; and (e) repeating steps (c) through (d) until all of the peripheral blood samples are taken The cells are collected into the desired cell population. In certain embodiments, the first flow rate is 20 ml/min or 20 ml/below red and the first target cell population comprises platelets. In certain embodiments Increasing the flow rate to 50 ml/min or 50 ml/min or less 152717.doc • 17·201130978 and the target cell population contains a minimal embryonic-like (VSEL) stem cell population β. In some embodiments, the increased flow rate is 70. In milliliters per minute or below 7 milliliters per minute. In some embodiments, the increased flow rate is 90 milliliters per minute or less and the target population of cells comprises red blood cells (rbc) and white blood cell (WBC) populations. In certain embodiments, the increased flow rate is greater than 9 〇ml/min or less than 90 cc/min and the target population of cells comprises a population of hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). In certain embodiments, The increased flow rate is about 105 milliliters per minute or less than 1 milliliter per minute, and the target population of cells comprises a population of hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). In certain embodiments, the methods disclosed herein Do not Forward selection techniques, such as immunoselection or immunological density selection, such as FACS. In certain embodiments, the methods disclosed herein do not use a positive selection technique that utilizes antibodies or antibody fragments. In certain embodiments, Negative selection techniques are applied to the population of cut-off cells to enrich the desired population of target stem cells in the population of cut-off cells. In certain embodiments, the negative selection technique uses immunoselection and immuno-dense selection. In certain embodiments, Negative selection techniques are used to remove undesired cells and components from a target stem cell population. In certain embodiments, the method can be used to derive from peripheral blood samples (such as human peripheral blood samples, including mobilized peripheral blood samples). A plurality of stem cell populations are obtained. In some embodiments, the peripheral blood sample is obtained from a human entity after the dispenser, for example, wherein the mobilizer has been administered to a human subject until 1 day. In one embodiment of the invention, the mobilizer is administered for two days. In another embodiment, the mobilizer is administered for three days. In some; 152717.doc 201130978, the peripheral blood sample is a separation product. Another aspect of the invention relates to a method of treating a disease or condition in an individual with a population of at least a solid autologous stem cell comprising: (a) utilizing at least one stem cell population from a peripheral blood sample, wherein the week "(4) self Injecting the transfer agent for individuals of 4 days or less, and the pot is enriched by at least one using a flow rate separation method without using a positive selection technique.

The stem cell population Kb) is administered to the individual to treat at least one stem cell phase I disorder. 3 In certain embodiments, at least one stem cell population obtained using the methods disclosed herein has been cryopreserved. In certain embodiments, at least one stem cell population comprises non-stem cells, such as a population of stem cells comprising at least 1 (%) of the target stem cell population. In certain embodiments, at least __ stem cell populations are substantially pure populations of target stem cell populations. In certain embodiments, at least one stem cell population has been expanded in vitro prior to administration to an individual, such as a human individual. In a certain embodiment, the method provides for collecting a plurality of stem cell populations from peripheral blood, wherein at least one stem cell population is a minimal embryonic-like (VSEL) stem cell, and at least one stem cell population is MSC, and at least one stem cell population is in some implementations In one embodiment, the at least one stem cell population administered to the individual is a combination of cells comprising any combination selected from the group consisting of minimal embryonic (VSEL) stem cells, MSCs or HSCs. In certain embodiments, the invention provides a method of isolating a population of target cells from peripheral blood, comprising: (a) flowing a peripheral blood sample from the individual through a fluid chamber of the panning apparatus at a first flow rate, The first flow rate is selected such that cells of the peripheral blood 152717.doc -19- 201130978 liquid sample are smaller than the target cell population flow through the fluid chamber, and the target cell population is trapped in the fluid chamber; (b) first The flow rate is increased to a second flow rate to cause the target cell population to flow through the fluid chamber; and (c) the target cell population is collected as the target cell population flows through the fluid chamber; wherein steps (a), (b), and (c) Does not include the use of positive selection techniques. In certain embodiments '(0 selects a first flow rate such that cells in the peripheral blood sample that are smaller than minimal embryonic stem cells (VSEL) flow through the fluid chamber and VSEL is trapped in the fluid chamber' and (Π) select The second flow rate is such that the VSEL flows through the fluid chamber. In certain embodiments, (1) the first flow rate is about 35 ml/min, the second flow rate is about 50-70 ml/min, and the VSEL is collected. In some embodiments, '(1) selects a first flow rate such that cells of less than mesenchymal stem cells (MSC) in the peripheral blood sample flow through the fluid chamber and the MSC is trapped in the fluid chamber' and (ii) selects a second flow rate to The MSC is allowed to flow through the fluid chamber. In certain embodiments, (1) the first flow rate is about 10 〇 ml/min, the second flow rate is about 110-120 ml/min, and the MSC is collected. (i) selecting a first flow rate such that cells in the peripheral blood sample that are smaller than hematopoietic stem cells (HSC) flow through the fluid chamber and the HSC is trapped in the fluid chamber, and (ii) selecting a second flow rate to cause the HSC Flow through the fluid chamber. In some embodiments '(1) first flow rate The rate is about 9 〇ml/min, the first rate of motion is about 100 ml/min, and the HSC is collected. 152717.doc • 20· 201130978 In certain embodiments, the positive selection technique is immune selection or immune density selection. In certain embodiments, the peripheral blood sample is a human peripheral blood sample. In some embodiments, the peripheral blood sample is a mobilized peripheral blood sample. In certain embodiments, the peripheral blood sample is obtained from a administered mobilizer 4 曰 or individuals below 4 days. In certain embodiments, the collected target cell population is cryopreserved. In certain embodiments, at least one of the collected target cell populations is administered to the individual. In certain embodiments At least one collected target cell population is initially administered to an individual who initially provides peripheral blood. In certain embodiments, at least one of the collected target cell populations has been expanded in vitro prior to administration to the individual. In certain embodiments The individual is a human individual. In certain embodiments, at least one of the collected target cell populations is a VSEL. In certain embodiments, at least one of the collected target cell populations is an MSC. In some embodiments, at least one of the collected target cell populations is an HSC seed from a peripheral blood separation target. In certain embodiments, the present invention provides a method of cell population comprising: (a) The liquid sample flows through the fluid chamber of the 152717.doc -21·201130978 panwashing device at a first flow rate, wherein the first flow rate is selected such that the first target cell population flows through the fluid chamber and is collected, And the first residue of the surrounding sputum sample is trapped in the fluid chamber; and (b) increasing the first flow rate to a second flow rate such that the second target cell population flows through the fluid chamber and is collected The _th residue of the blood sample is trapped in the fluid chamber; wherein steps (a) and (b) do not include the use of positive selection techniques. In certain embodiments, the method further comprises: (c) increasing the second flow rate to a third flow rate such that the third mesh, the population of cells flow through the fluid chamber and is collected, and the peripheral blood sample is The three residues are trapped in the fluid chamber; wherein step (c) does not involve the use of a forward selection technique. In certain embodiments, the method further comprises: (d) increasing the third flow rate to a fourth flow rate such that the fourth target population of cells flows through the fluid chamber and is collected, while the fourth remaining of the peripheral blood sample The material is trapped in the fluid chamber; wherein step (d) does not involve the use of a forward selection technique. In certain embodiments, the method further comprises: (e) increasing the fourth flow rate to a fifth flow rate such that the fifth target cell population flows through the fluid chamber and is collected, and the peripheral blood sample is The five residues are trapped in the fluid chamber; wherein step (e) does not involve the use of positive selection techniques. In certain embodiments, the method further comprises: (f) increasing the fifth flow rate to a sixth flow rate such that the sixth target 152717.doc • 22· 201130978 cell population flows through the fluid chamber and is collected, The sixth remainder of the peripheral blood sample is trapped in the fluid chamber; wherein step (f) does not involve the use of positive selection techniques. In certain embodiments, the method further comprises: (g) increasing the sixth flow rate to a seventh flow rate such that the seventh target population of cells flows through the fluid chamber and is collected, while the seventh remaining of the peripheral blood sample The material is trapped in the fluid chamber; wherein step (g) does not include the use of positive selection techniques. In certain embodiments, the first flow rate is selected such that the first target population is less than minimal embryonic stem cells (VSEL). The cells, and (ii) select a second flow rate such that the second target population of cells is a VSEL. In certain embodiments, (i) the first flow rate is about 35 ml/min, the first flow rate is about 50-70 ml/min, and the VSEL is collected. In certain embodiments, (1) selecting a third flow rate such that the first target population is less than cells of hematopoietic stem cells (HSC), and (π) selecting a fourth flow rate such that the second target population is HSC. In certain embodiments '(i) the third flow rate is about 9 〇mi/min, the fourth flow rate is about 100 ml/min, and the HSC is collected » In some embodiments '(1) selects the fourth flow rate The first target cell population is a cell that is smaller than the mesenchymal stem cells (MSC), and the fifth flow rate is selected such that the second target cell population is the MSC. In certain embodiments, '(1) a third flow rate of about 10 〇 ml/min, a fourth flow rate of about 110-120 ml/min, and collecting MSCs. In certain embodiments, '(i) a first flow rate of about 35 ml/min and a 152717.doc -23. 201130978 target cell population is platelets; (ii) a second flow rate of about 50 ml/min and The second target cell population is VSEL; (iii) the third flow rate is about 70 ml/min and the third target cell population is VSEL; (iv) the fourth flow rate is about 90 ml/min and the fourth target cell population is Red blood cells; (v) a fifth flow rate of about 100 ml/min and a fifth target cell population of HSC; (vi) a sixth flow rate of about 110 ml/min and a sixth target cell population of MSC; and (vii) The seventh flow rate is about 120 ml/min and the seventh target cell population is MSC. In some embodiments the 'forward selection technique is immunoselection or immune density selection. In certain embodiments, the peripheral blood sample is a human peripheral blood sample. In some embodiments, the peripheral blood sample is a mobilized peripheral blood sample. In some implementations, the peripheral blood sample is obtained from an individual who has administered the mobilizer for 4 or less days. In certain embodiments, the collected target cell population is cryopreserved. In certain embodiments, at least one of the collected target cells administered to the individual, in certain embodiments, is administered at least one of the target cell populations collected from the individual providing the peripheral blood at least one of 152717.doc -24 - 201130978. In some embodiments, at least one of the makeup nh Μ P J-in, the target cell population that has been scared to collect has been expanded in vitro prior to administration to the individual. In certain embodiments, the individual is a human individual. _ In some embodiments, at least one of the collected target cell populations is • VSEL. In some embodiments, at least one of the target cell populations collected is MSC. In certain embodiments, at least one of the collected target cell populations is an HSC. In certain embodiments, the present invention provides a method of isolating a target cell population from peripheral blood, comprising: (4) selecting a peripheral blood sample from the individual to flow through a fluid chamber of the panning apparatus at a first flow rate a first flow rate such that the first target population of cells flows through the fluid chamber and is collected while the first residue of the peripheral blood sample is trapped in the fluid chamber; (b) increasing the first flow rate to the second flow rate So that the second target population of cells flows through the fluid chamber and is collected, while the second remainder of the peripheral blood sample is trapped in the fluid chamber; and (c) repeating step (b) as appropriate until the desired number is collected Target cell population; wherein steps (a), (b), and (c) do not include the use of positive selection techniques. In certain embodiments, the invention provides a VSEL, MSC or HSC product by a method as disclosed above. 152717.doc • 25- 201130978 [Embodiment] In one embodiment, the present invention achieves the flexibility of optimizing the recovery of a specific target stem cell population from peripheral blood samples, wherein the enrichment of a plurality of different target stem cell populations is high in yield. A number of cells with low cross-cell contamination of non-stem cells or non-target stem cells. One aspect of the present invention relates to the rapid and reliable separation of bulk stem cell populations from peripheral blood using a panning method, wherein each target stem cell population has a different Different sedimentation coefficients (size and density) of the target stem cell population, thereby enriching different target stem cell populations, wherein the recovery or viability of the target stem cell population is almost no loss. In certain embodiments, target stem cells obtained from peripheral blood according to the methods disclosed herein are suitable for use in cell therapy. In some stem cell therapies, bone marrow (BM) or peripheral blood (PB) derived stem cells are used for cell replacement or regenerative therapy. Methods for isolating stem cells and non-stem cells can be divided into three basic types: (a) separation based on cellular physical properties (such as cell density and size), including fluid flow (field flow fractionation) or sedimentation field (centrifugation) or A method of differential migration of fluid flow (panning) in the presence of a settling field. The differential sinking can be combined with a solution or hydrogel of different intrinsic density in a discontinuous or continuous (gradient) form. (b) Separation based on cell surface characteristics such as unit charge (electrophoresis) or interfacial free energy (phase portion) or the presence of specific surface molecular groups (interacting with various affinity surfaces). Examples of separation based on specific surface molecular groups include the use of cell surface receptors or other determinants of affinity 152717.doc -26- 201130978 force single or multiple antibodies. Affinity methods include negative selection (eg, interaction with cell surface receptors and affinity monoclonal antibodies to remove undesired cell populations) or positive selection (interacting with cell surface receptors and affinity molecules (eg, monoclonal antibodies) To select the desired target cells). (C) Selection based on a combination of cellular physical properties and surface characteristics, e.g., based on a combination of a density gradient medium for isolating a population of cells and a cell surface specific antibody that recognizes a target cell or an undesired subset of cells. However, 'stem cell isolation is usually based on cell surface characteristics. In addition, conventional methods for isolating stem cell populations (such as HSCs, MSCs, and VSELs) from peripheral blood samples typically include cell surface-based separation methods, such as immunoselection, in which isolation and isolation are specific. The stem cell population is based on the binding of affinity molecules (such as antibodies or cell surface ligands) to the surface of the target stem cells to be isolated. Such specialized separation methods are generally known in the art and include, without limitation, fluorescent cell sorting (FACS), immunodensity separation, and immunomagnetic separation, and the like. Therefore, conventional methods for isolating different stem cell populations from peripheral blood require the contamination of surrounding j^L liquid samples and are expensive and slow, and often require specialized training and/or specialized equipment by skilled professionals or technicians. In addition, conventional methods often only enrich one stem cell population in a peripheral blood sample and discard other potentially unselected or unenriched stem cell populations present in the peripheral blood sample. In addition, there are problems in conventional methods for enriching stem cells from peripheral blood, such as contamination of other non-target stem cells, which can interfere with various cell isolation and/or cell selection techniques and the subsequent selection of target stem cells for therapeutic use. Therefore, it is necessary to separate stem cells from peripheral blood into a population of stem cells. A subset is required and the number of non-target stem cells in each subset of stem cells is removed or reduced. The present invention provides a method of isolating a desired population of stem cells which can be carried out using a large number of input cells without the use of a cell surface marker or a method of selection of the agent associated with the method. The present invention provides separation parameters and demonstrates that several types of stem cells can be effectively enriched from peripheral and other cell components that are separated from the blood. In certain embodiments, the resulting portion comprising the target stem cell population can be further separated into a desired subset of cells for collection if necessary. It has previously been reported that separation of G-CSF by countercurrent centrifugation elutes PBSC grafts to moderately thicken CD34+ cells without loss of primary hematopoietic progenitor cells (see Kwekkeboom et al., British Journal of Haematology, 1997, 99; 47). -55). Kwekkeboom et al. discuss the use of countercurrent centrifugal elution (CCE) to reduce the number of non-dry sacs in autologous G-CSF mobilized peripheral blood stem cell (PBSC) grafts. The hematopoietic progenitor cells present in the small cell and leukapheresis product (LP) samples can be rapidly separated by cell size-monitored CCE. However, unlike the present invention, Kwekkeboom et al. do not discuss the use of CCE to isolate different stem cell populations from peripheral blood samples. More specifically, Kwekkeboom did not find an indication that the primary stem cells were preferentially washed with any part (including the small cell fraction).

Micklethwaite et al. discuss the clinical scale elution of monocytes using ELUTRA® to enrich the immunotherapy regimen, and the analysis of four (F1-F4) non-monocyte fractions derived from this method, and discuss ELUTRA® Enrichment can be incorporated into and enhances existing immunotherapy and stem cell transplantation 152717.doc • 28-5: 201130978 Cell subsets of the program (Micklethwaite et al., C/zwica/jca/e elutriation as a means of enriching antigen-presenting Cells and manipulating alloreactivity, Cytotherapy; 2009, 11; 218-228). Micklethwaite et al. discuss the use of FACS to analyze T, B, natural killer (NK) and dendritic cells (DC) from different ELUTRA® fractions (such as F3-F4). However, unlike the present invention, Micklethwaite et al. discuss the use of ELUTRA® to mobilize hematopoietic cells, such as dendritic cells or natural killer cells, from human peripheral blood, rather than to thicken stem cell populations. For the sake of convenience, certain terms used in the specification, examples, and accompanying claims are hereby incorporated herein. Unless otherwise stated or implied by the context, the following terms and phrases include the meanings provided below. Unless otherwise expressly stated or apparent from the context, the following terms and phrases § I do not exclude terms or phrases that have been obtained in the art to which they belong. The present invention is provided to facilitate the description of the specific embodiments and is not intended to limit the invention. This is because the scope of the invention is limited only by the scope of the patent application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning meaning As used herein, the term "apheresis" refers to a method or procedure for drawing blood from a donor individual and isolating it into its components, some of which are truncated, such as plasma, platelets, and/or stem cell populations, and remaining This method can also be referred to as blood transfusion in the art to return to the donor individual. The form of separation includes blood: leukocyte separation-heavy-heterogeneous or pheresis. Pulp separation - collecting blood damage (liquid part of the blood) 152717.doc -29· 201130978 white blood cells; granulocyte separation (Granulocytapheresis) - collecting granulocytes (neutrophils, eosinophils and eosinophils) Lymphocytapheresis-collecting lymphocytes; lymphocyte plasmapheresis (Lymphoplasmapheresis) - collecting lymphocytes and blood pooling; platelet detachment (Plateletpheresis; thrombocytapheresis) - collecting platelets. The separation of blood is longer than the whole blood donation. It takes about 10-20 minutes for blood to collect blood, and apheresis donation can take about 1-2 hours. As used herein, sS "separation product" refers to a heterogeneous population of cells collected by a method of separation. As disclosed herein, cells present in the isolated product can be isolated by panning. As used herein, the term "panning" refers to the separation or enrichment of cells based on their differential sedimentation rate, such as stem cells from peripheral blood. The panning method used in this paper is a non-invasive separation of a large number of cells based on their size and quality! Method of birth. Again, the panning process allows for the isolation of mixed cells (especially stem cells at different stages of the cell division cycle) without interfering with stem cell metabolism or using a synchronizing agent. The terms "enriching" or "enriched" or "enrich" are used interchangeably herein and mean the yield (ie fraction) of a type of cell compared to the starting culture. Or the amount of cells of this type is increased by at least 10%. » The term "separation" or "selection" as used herein refers to the separation of different cell types into - or multiple groups and collection of isolated populations as a concentration in specific target stem cell populations. m cell group. You can choose between positive selection (for thickening I52717.doc 201130978 cells) or negative selection (disposal of non-target cell populations). As used herein, the term "purification" means the separation of undesired components. As used herein, the term "positive selection" refers to, for example, a method of targeting a desired stem cell for selection using a monoclonal antibody of a specific cell surface antigen on a desired or target stem cell. As used herein, the term 'negative selection' refers to, for example, the use of a monoclonal antibody filament of a specific cell surface antigen to an undesired or non-target stem cell in order to eliminate the negative selection of the sputum, and the desired cell or target cell is not labeled with an antibody such as 0. As used herein, the term 'removal' means separating and selecting and leaving (9), retaining or disposing of it. Therefore, the target cell population can be removed from the mixed cell population in order to retain it or discard it. As used herein, the term "immunoselection" refers to a method of labeling cells (e.g., stem cells) using a monoclonal antibody and selecting antibodies by using a label (such as fluorescent or magnetic particles) on the antibody. As used herein, the term "immune density selection" refers to a method of dry-to-unwanted cells (e.g., non-target cells) with a single antibody-based reagent and agglomerate when centrifuged through a density medium. As used herein, the term "fractionation" refers to the generation of a homologous group of stem cell populations from a heterogeneous stem cell population. As used herein, the term "peripheral blood" refers to whole blood obtained from an individual. As used herein, the term 'transferred peripheral blood' refers to a peripheral fluid obtained from an individual in which the individual mobilizer has been administered to increase the number of stem cells in the peripheral blood' by increasing the stem cells from the bone marrow (eg, increasing the source) 152717.doc -31 - 201130978 Stem cells) migrate to peripheral blood' or increase the proliferation of stem cells present in peripheral blood (eg increase the number of PB-derived stem cells). The mobilization can be achieved in an individual by administering an effective amount of a mobilizing agent, such as a combination of a chemical attractant (e.g., a cytokine) and loss of adhesion to a pool or population of stem cells in peripheral tissue and stem cell niches in the bone marrow. An "effective amount" is an amount of a mobilizing agent sufficient to achieve 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, administrations or administrations. The therapeutically effective amount of the mobilizing agent will depend on the selected mobilizing agent (e.g., G_CSF or GM-CSF). A mobilizing agent such as g-CSF or GM-CSF can be administered from one or more times per week to one or more times per week (including once every other day). Those skilled in the art should be aware that 'certain factors may affect the dosage and timing required to effectively treat an individual', including but not limited to prior treatment, general health and/or age of the individual, and whether other disease. Further, as is generally known to those of ordinary skill in the art, treating a subject in a therapeutically effective amount with G-CSF or GM-CSF can include a single treatment or a series of treatments. As used herein, the term "mobilized" refers to a method of leaving cells out of the bone marrow and into the blood. The mobilization can be achieved by combining chemical bowing agents (e. g., cytokines) and loss of adhesion of stem cell pools or populations present in peripheral tissue and stem cell niches in the bone marrow. The term "stem cell" as used herein is used broadly and includes conventional stem cells, tendon cells, prepr0genit〇r cells, reserve cells, and the like. The term "stem cell" or "progenitor cell" is used interchangeably herein and refers to the ability to proliferate and produce more 152717.doc • 32·5 which have a large number of blasts that can produce differentiated or differentiated daughter cells. . 201130978 Undifferentiated cells of the cell. The daughter cells themselves can be induced to proliferate and produce progeny, which then differentiate into one or more (four) mature cell types, while also retaining one or more cells with parental developmental potential. The term "dry cell" refers to a cell that, under certain circumstances, has the ability to differentiate into a more specific or differentiated phenotype, and in some cases retains the ability to proliferate without substantial differentiation. In one embodiment, the term progenitor or stem cell refers to a generalized mother cell whose progeny (progeny) are often differentiated in different directions, such as in embryonic cells and tissues, by obtaining fully individual features, for example. It occurs in the progressive diversification. Cells differentiate into complex processes that typically occur through many cell divisions. The differentiated cells can be derived from pluripotent cells derived from pluripotent cells or the like. Although each of these pluripotent cells can be considered a stem cell', the range of cell types that can be produced by each can vary considerably. Some differentiated cells also have the ability to produce cells with greater developmental potential. This ability can be natural or can be artificially induced when treated with various factors. In many cases, stem cells are also "pluripotent" because they produce offspring of more than one different cell type, but this is not required for "stem-ness". Self-renewal is the other classic part of stem cell definition and is required as used in this document. In theory, self-renewal can be wrong by either of the two main mechanisms. Stem cells can divide asymmetrically, with one progeny retaining the stem cell state and the other progeny exhibiting some other specific functions and phenotypes. Alternatively, some of the stem cells in the population can be symmetrically split into two stem cells, thus maintaining some of the stem cells in the population as a whole, while the other cells in the population only produce differentiated progeny. Formally, cells starting with stem cells may proceed to a differentiated phenotype, but then “reverse 152717.doc • 33· 201130978 and then reproduce the stem cell phenotype (often referred to as the term “anti-differentiation”). 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, cartilage stem cells, lymphoid stem cells, mesenchymal stem cells, hematopoietic stem cells, central nervous cells. System stem cells, peripheral nervous system stem cells and the like. Description of stem cells, including their isolation and culture methods, can be found (among other places) Embryonic Stem Cells, Methods and Protocols, Turksen, ed., Humana Press, 2002; Weisman et al, Annu Rev. Cell. Dev. Biol. :387 403; Pittinger et al, Science, 284:143 47, 1999; Animal Cell Culture, Masters, Oxford University Press, 2000; Jackson et al, PNAS 96(25): 14482 86, 1999; Zuk et al, Tissue Engineering, 7: 211 228, 2001 ("Zuk et al."); Atala et al., especially Chapter 33 41; and U.S. Patent Nos. 5,559, 〇 22, 5, 672, 346 and 5, 827, 735. Description of stromal cells (including methods for their isolation) can be found (among other places) prock〇p, Science, 276: 71 74, 1997; Theise et al, Hepatology, 31: 235 40, 2000; Current Protocols in Cell Biology, Edited by Bonifacino et al., j〇hn

Wiley & Sons, 2000 (including updates to March 2002), and U.S. Patent No. 4,963,489. The term "progenitor cell" is used herein to mean a stem cell that has a more primitive cell phenotype relative to cells that can be produced by differentiation (e.g., along a developmental pathway or progression, at an earlier step than a fully differentiated cell). Progenitor cells often also have significant or extremely high proliferative potential. Depending on the developmental pathway and cell development and differentiation 152717. Doc 5.  201130978 Environment-dependent 'progenitor cells can produce multiple different differentiated cell types or single differentiated cell types. As indicated above, there are different degrees or categories of cells that are generally derogatory to "stem cells." It is "totipotent", "plurip〇tent" and "multipotent" stem cells. The term "all-round" refers to stem cells that produce any tissue or cell type in the body. "Reenergy" stem cells can produce any type of cells other than germline cells in the body. Stem cells that produce smaller or limited numbers of different cell types are commonly referred to as "multiple months b." Thus, totipotent cells differentiate into multipotent cells that produce most, but not all, tissues necessary for fetal development. Recombinant cells undergo further differentiation into pluripotent cells that are committed to produce cells with specific functions. For example, pluripotent hematopoietic stem cells produce red blood cells, white blood cells, and platelets in the blood. As used herein, the term "repotential" refers to a cell that has the ability to differentiate into cell types unique to all three germ cell layers (endoderm, mesoderm, and ectoderm) under different conditions. The pluripotent cells are characterized mainly by their ability to differentiate into all three germ layers using, for example, a nude mouse teratoma formation assay. It is also confirmed by the expression of embryonic stem cell (ES) cell markers that the better test is to demonstrate the ability to differentiate into cells of the three germ layer 夂ό 0 cents. In certain embodiments, the potent cells are undifferentiated cells. As used herein, the term "re-energy" or "re-energy state" refers to differentiation into all three embryonic germ layers: endoderm (ssue), mesoderm (including blood, muscle and blood vessels), and ectodermal growth. , 61, such as the skin and nerves, and usually have the potential to divide in vitro for a long period of time (eg, greater than or greater than 30 passages). 'year 152717. Doc •35· 201130978 When used with reference to “pluripotent cells,” the term “pluripotent” refers to cells that can be differentiated into cells that are derived from some, but not all, of all three germ layers. Therefore, pluripotent cells are partially differentiated cells. Pluripotent cells are well known in the art and examples of pluripotent cells include adult stem cells, such as hematopoietic stem cells and neural stem cells. Multi-capable means that stem cells can form many types of cells in a given line, rather than other lineage cells. For example, pluripotent blood stem cells can form many different types of blood cells (red, white, platelets, etc.), but they cannot form neurons. The term "pluripotent" refers to cells whose development versatility is less than that of omnipotent and complex energy. The term "total energy" refers to a cell having a degree of differentiation that describes the ability to produce all cells in adult and extraembryonic tissues, including the placenta. Fertilized egg (zygote) is omnipotent because it is an early lytic cell (blastomere). The term "mesenchymal stem cell" is also referred to herein as "MSC" and refers to the ability to differentiate into more than one specific type of leaf tissue or connective tissue (ie, to support specialized elements; eg, fat, bone Complex cells of the matrix, cartilage, elasticity, and tissues of the fibrous connective tissue. Human mesenchymal stem cells (hMSCs) are reactive with certain monoclonal antibodies, such as SH2, SH3, and SH4 (see U.S. Patent No. 5,486,359, the disclosure of which is incorporated herein by reference in its entirety herein The specific pattern is distinguished from HSC and wherein MSC is SH2+/CD14- and human HSC SH2-/CD14+. For identification purposes, human MSC can be identified based on: (i) CD34-, CD45-, CD90+, CD105+ And 152717. Doc • 36- 5 201130978 CD44+ phenotypic marker expression, (Π) functional phenotype, including the ability to form colony forming units in CFA assays as disclosed in the examples herein, and differentiation into supportive specific elements (including (but not Limited to: the ability to organize tissues of chondrocytes, cartilage and fat cells. Other markers represented by MSC are known in the art and include, without limitation, CD71, CD73, Stro-Ι, and CD166, and CD271. In certain embodiments, the MSC is lin-. The term "very small embryonic stem cells" is also referred to herein as "VSEL stem cells j and refers to pluripotent stem cells. In certain embodiments, VSEL stem cells ("VSEL") are human VSELs and can be characterized as lin·, CD45· And CD34+. In certain embodiments, the VSEL is a human VSEL and can be characterized as lin·, CD45·, and CD133+. In certain embodiments, the VSEL is a human VSEL and can be characterized as lin·, CD45·, and CXCR4+. In certain embodiments, the VSEL is a human VSEL and can be characterized as lin·, CD45·, CXCR4+, CD133+, and CD34+. The human VSEL exhibits at least one of SSEA-4, Oct-4, Rex-1, and Nanog, and has a large nucleus surrounded by a narrow side of the cytoplasm and contains embryonic unorganized chromatin. VSEL also has high-end granzyme activity. In certain embodiments f, the VSELs are human VSELs and can be characterized as lin·, CD45_, CXCR4+, CD133+, Oct 4+, SSEA4+, and CD34+. In certain embodiments, 'human VSELs may be less primitive and may be characterized as lin·, CD45·, CXCR4+, CD133·, and CD34+. In certain embodiments, a human VSEL can be enriched for a reassortant embryonic transcription factor (e.g., 〇ct_4, s〇x2, and Nanog). In some embodiments, the diameter of the human VSEL can be 4-5 μηι, 4-6 μηη, 4-7 μηη, 5-6 μηι, 5-8 μπι, 6-9 μηι or 7·10 μιη 〇 152717. Doc •37- 201130978 The term “peripheral blood source” used in connection with stem cells refers to stem cells that are only mobilized from peripheral blood and may include stem cell expansion or proliferation in peripheral blood. In certain embodiments, according to the methods disclosed herein, the number of circulating stem cells in the peripheral blood can be increased by contacting the peripheral blood with the mobilizing agent in vivo or ex vivo. The term "bone marrow source" as used in connection with stem cells refers to stem cells that are mobilized from bone marrow to peripheral blood' and may include stem cells that have migrated from BM. In certain embodiments, BM-derived stem cells in peripheral blood include stem cells that have proliferated in the bone marrow prior to migration into peripheral blood, or stem cells that have been proliferated in peripheral blood after migration from bone marrow. In certain embodiments, the number of circulating BM stem cells in the peripheral blood can be increased by contacting the peripheral fluid with the mobilizing agent in vivo according to the methods disclosed herein. Also known as "HSC", "hematopoietic stem cells" refers to the ability to differentiate into any particular type of hematopoietic or blood cells (such as red blood cells, lymphocytes, macrophages, and megakaryocytes), especially in the bone marrow and peripheral blood. All stem or progenitor cells. HSCs are reactive with certain monoclonal antibodies (e.g., monoclonal antibodies that recognize CD34) that are now recognized to be specific for hematopoietic cells. As will be understood by those skilled in the art, the term "hematopoietic cells" refers to all types of hematopoietic cells throughout their differentiation from self-renewing hematopoietic stem cells to immature precursor cells of various blood systems, including mature acting blood cells. Sf. "mesenchymal cells" or "mesenchyelia" are used interchangeably herein and in some instances refers to spindle-like or stellate cells found between the ectodermal and endoderm of immature embryos. Most mesenchymal cells are derived from established mesoderm, but in the head 152717. Doc -38 · 201130978 It also develops from the nerve ridge or neural tube ectoderm. Mesenchymal cells (specifically, embryonic mesenchymal cells in embryonic bodies) have recombination ability, which develops into any type of connective or supporting tissue of smooth muscle, vascular endothelium and blood cells at different locations. As used herein, the term "isolated cell" refers to a cell that has been removed from an individual that was originally seen or a progeny of such a cell. Optionally, the cells are cultured in vitro, for example, in the presence of other cells. Optionally, cells (eg, a population of isolated target stem cells produced by the methods disclosed herein) are introduced into the second individual or reintroduced into the individual (eg, allograft) of the individual (or passaged cells) )in. As used herein, with respect to isolated cell populations, the term "isolated population" refers to a population of cells that have been removed and isolated from a mixed or heterologous population of cells. In certain embodiments, the isolated population is a substantially pure population of cells as compared to a heterologous population of cells that are thereby isolated or enriched. In certain embodiments, the isolated population is a population of isolated reprogrammed cells that are substantially purely re-compared to a heterogeneous population of cells comprising reprogrammed cells and cells that produce reprogrammed cells. The programmed cell population. The term "substantially pure" with respect to a particular target stem cell population means that the cell population is at least about 75%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95, relative to the cells that make up the total stem cell population. %pure. Reaffirming that the term "substantially pure" or "substantially purified" with respect to a target stem cell population isolated using the methods disclosed herein means that the target stem cell population contains less than about 20%, more preferably less than about 15%, 10%. , 8%, 7%, optimally less than about 5%, 4%, 3%, 2%, 1% or less than 1% as defined by the terminology of 152717. Doc •39- 201130978 Cells of non-target stem cell populations. In certain embodiments, the invention encompasses a method of expanding a population of target stem cells wherein the target population of expanded stem cells is a substantially pure target population of stem cells. As used herein, "proliferation" refers to increasing the number of cells in a population (growth) by means of cell division. Cell proliferation is generally thought to be caused by coordinated activation of multiple signal transduction pathways that are responsive to the environment, including growth factors and other cleavage materials. Cell proliferation can also be promoted by relieving the effects of intracellular or extracellular signals and blocking or negatively affecting cell proliferation. The term "regeneration" means that a cell population, organ or tissue grows after a disease or trauma. The terms "update" or "self-renewal" or "proliferation" are used interchangeably herein and refer to the process by which a cell produces more copies of itself (eg, cell replication). In certain embodiments, the reprogrammed cells can be renewed by me by long-term and/or splitting into the same undifferentiated cells (e.g., pluripotent or non-specific cell types) over many months to years. In some cases, proliferation refers to the expansion of reprogrammed cells by repeated splitting into two identical daughter cells by a single cell. As used herein, the term "lineage" refers to a cell having a common ancestry or a cell having a common developmental fat, such as a cell derived from the same target stem cell population or a progeny thereof. As used herein, the term "pure cell line" refers to a cell line that can be maintained in culture and has the potential to immortalize. The pure cell line may be a stem cell strain (e.g., a target stem cell population cell line) or derived from a target stem cell population, and a pure line 152717 is used in the case of a pure cell line containing the target stem cell population. Doc -40· 5.  201130978 Cell line' The term refers to a population of target stem cells that have been cultured under in vitro conditions that allow proliferation without differentiation for months to years. Such pure stem cell lines (e. g., target stem cell populations) may have the potential to differentiate from primitive stem cells following several cell lines. "In cell individualogenesis, the adjective "differentiation" is a relative term. A "differentiated cell" is a cell that has progressed further down the developmental path than the cell to which it is compared. Because, 匕, stem cells can differentiate into lineage-restricted precursor cells (such as mesoderm stem cells), which in turn can further down-divide into other types of precursor cells (such as cardiomyocyte precursors) along the pathway, and then differentiate into end-stage differentiated cells, It has a characteristic role in a certain tissue type, and "T flb or may not maintain the ability to further proliferate. The term "differentiation" in the context of the present invention means the formation of a cell which exhibits a marker associated with a cell which is known to be (4) alienated and which is close to a terminally differentiated cell which cannot be further differentiated. The progression of cells from a less defined cell to a cell that is increasingly committed to a particular cell type, and ultimately to a terminally differentiated cell, is referred to as progressive differentiation or progressive typing. Cells that are more specific (e.g., have progressed along the progressive differentiation pathway) but have not yet been terminally differentiated are referred to as partial differentiation. Differentiation is a developmental process whereby the cells exhibit a specific phenotype, such as obtaining - or a variety of features or functions different from other cell types. In the case of one or two conditions, a differentiated phenotype is a cell phenotype (so-called terminally differentiated cell) at the mature end point in some developmental pathways. In many, but not all, tissues, the differentiation process is accompanied by a σ in the cell cycle. In such cases, the terminal divides the cell loss or greatly limits its ability to proliferate. However, we note that in the context of this specification, the term "differentiation" refers to the fate or merit in its I527I7. Doc •41– 201130978 Cells that are more specific in their energy than their pre-developmental points, and include terminally differentiated cells and cells that are more specialized than their previous point of development, although they are not terminally differentiated. Cells develop from unshaped cells (e.g., stem cells) to cells of a particular differentiated cell type with increased degree of conformation, and eventually develop into terminally differentiated cells. This is called progressive differentiation or progressive typing. Cells that "differentiate" relative to progenitor cells have one or more phenotypic differences relative to the progenitor cells. Phenotypic differences include, but are not limited to, morphological differences and differences in gene expression and biological activity, including not only the presence or absence of performance markers, but also differences in the amount of labeling and differences in the co-expression patterns of a panel of markers. As used herein, the term "differentiation" refers to the development of cells from the original stage to cells that are more mature (i.e., less primitive) cells. As used herein, the term "directed differentiation" refers to forcing cells to differentiate from undifferentiated (e. g., more primitive cells) to more mature cell types (i.e., less primitive cells) via genetic and/or environmental manipulation. In certain embodiments, cells reprogrammed as disclosed herein are directed to differentiate into specific cell types, such as neuronal cell types, myocyte types, and the like. As referred to herein, the term "medium" is a medium that maintains a tissue or population of cells, or a population of cells (e.g., "medium") that contain nutrients that maintain cell viability and support proliferation. The cell culture medium may contain any of the following in a suitable combination: salts, buffers, amino acids, grape vinegar or other sugars, antibiotics, serum or serum substitutes, and other components such as peptide growth factors, and the like. Cell culture media commonly used for a particular cell type are known to those skilled in the art. The "phenotype" refers to one or a large number of general biological characteristics, which are in the environment 152717. Doc 5 •42· 201130978 Conditions and factors ♦ $ # $ κ 疋 景 景 定义 定义 定义 定义 定义 定义 定义 定义 定义 定义 定义 定义 定义 定义 定义 定义 定义 定义 定义 定义 定义 定义 定义 定义 定义As used herein, "marker" describes the characteristics and/or phenotype of a cell. It can be used to select cells that contain relevant features. The label should be characterized by the individual cells, ie the cell type or the morphological, functional or biochemical (enzymatic) characteristics characteristic of the molecule expressed by the cell type. Such labels are preferably proteins' and more preferably have antigenic epitopes of the antibody or other binding molecules known in the art. However, the label may consist of any knife found in the cell including, but not limited to, proteins (peptides and polypeptides), sorbitan, nucleic acids and steroids. Examples of morphological features or characteristics include, by definition, shape, size, and ratio of nuclear to cytoplasm. Examples of functional features or characteristics include, but are not limited to, the ability to adhere to or exclude a particular dye from a particular substrate, the ability to migrate under specific conditions, and the ability to differentiate along a particular line 4 line. The mark can be detected by any method available to those skilled in the art. As used herein, as used in connection with contacting a peripheral blood sample in vivo, the term "contacting" can include administering an individual mobilizing agent in the form of a composition, as appropriate, via a suitable route of administration such that the compound contacts the peripheral blood sample in vivo. As used herein, the term "contacting", as associated with ex vivo contact with a peripheral blood sample, can include, as the case may be, administering a mobilizing agent to the peripheral blood sample in the form of a composition such that the mobilizing agent contacts the peripheral blood sample ex vivo. As used herein, the terms "administering," "introducing," and "transplanting" are used interchangeably and refer to a method or pathway by which a human target stem cell population is at least partially localized to a desired site. The group is placed in a 152717. Doc •43· 201130978 In the body. The human target stem cell population can be administered by any suitable route that results in delivery to a desired location in the individual, wherein at least a portion of the human target stem cell population remains viable. The stage of cell survival after administration to an individual can be as short as a few hours, such as twenty-four hours to several days, up to several years. As used herein, the term "donor" is used to collect an individual to be transplanted an organ, tissue or cell. As used herein, the term "recipient" refers to an individual who will receive a transplanted organ, tissue or cell. As used herein, the term "transplantation" refers to a method of integrating free (unattached) cells, tissues or organs into a tissue after transplantation into an individual. § "Allografts" refers to transplanted cells, tissues or organs derived from different animals of the same species. As used herein, the term "Xen〇graft; xen〇transplant" refers to a transplanted cell, tissue or organ derived from an animal of a different species. In some embodiments, xenografting is the surgical transplantation of tissue from a species to a different species, genus or family. For example, transplanting from human to human is a xenotransplantation. "Heterotransplantation" refers to the method of transplanting living cells, tissues or organs from one species to another, such as from pigs to humans. The terms "subject" and "individua" are used interchangeably herein and refer to an animal, such as a human, from which it can be isolated and collected according to the methods and compositions described herein. The target stem cell population, and optionally the individual can be transplanted (eg, the target stem cell population can be implanted into the individual), for example, for treatment, including prophylactic treatment of the disease. For treatment to 152717. Doc 201130978 Special animal diseases (such as human individuals) have specific disease conditions, terminology. "Solid" means a specific animal. The term "non-human animal" Xiao "non-human ritual animal" is used interchangeably herein and includes mammals such as: mouse milk, rabbit, sheep, I seedling 'dog, cow, pig, and non-human primate. The π individual also covers any vertebrate, including (but not) mammals, reptile amphibians and fish. However, the individual is preferably a mammal, such as a human, or other mammal, such as a domestic mammal, such as a dog lick and the like, or a productive mammal, such as a cow, sheep, pig, and the like. The term "tissue" refers to a group or layer of similarly specialized cells that interact with certain functions. The term "tissue specificity" refers to the source or definition of the cellular characteristics of a particular tissue. As used herein, the term "agent" means any compound or substance such as, but not limited to, a small molecule, a nucleic acid, a polypeptide, a, a drug, an ion, and the like. "Pharmaceutical" can be any chemical substance, entity or part, including (without limitation) synthetic and naturally occurring proteinaceous and non-proteinaceous entities. In certain embodiments, the agent is a nucleic acid, a nucleic acid analog, a protein, an antibody, a peptide, an aptamer, a nucleic acid vesicle, an amino acid, or a carbohydrate, including, without limitation, oligo-acid, ribonuclease, DNAzymes, glycoproteins, siRNAs, lipoproteins, and modifications and combinations thereof. In certain embodiments, the agent is a small molecule having a chemical moiety. "For example, the chemical moiety includes an unsubstituted or substituted alkyl, aromatic or heterocyclic moiety, including a macrolide, a Lactobacillus. Uptomycin and related natural products or analogs thereof. The compounds are known to have the desired activity and/or properties, or may be selected from the collection of different compounds 152717. Doc •45- 201130978 Library. As used herein, the term "small molecule" refers to a chemical agent that may include, but is not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs. , aptamers, nucleotides, nucleotide analogs, organic or inorganic compounds having a molecular weight of less than about 10,000 g/mole (for example, including heteroorganic and organometallic compounds), having a molecular weight of less than about 5, 〇〇〇 克 /莫An organic or inorganic compound of the ear, an organic or inorganic compound having a molecular weight of less than about L000 g/mole, an organic or inorganic compound having a molecular weight of less than about 500 g/mol, and salts, esters, and other pharmaceutically acceptable forms. Special compound. The term "disease" or "condition" is used interchangeably herein and refers to any alternation of the state of the body or organs that disrupts or interferes with the effects of the function and/or causes the person being afflicted or contacted by the person. Symptoms, such as discomfort, dysfunction, pain or even death ^^ or illness can also be associated with dither (diStemper), ailing, ailment, disease (10) force, illness, disease, disease (Ulness) ), illness (c〇mpUint), discomfort (indisposition) or infection (affection). As used herein, the term "pathology" refers to a condition, such as a change in the structure and function of a cell, tissue or organ, which contributes to a disease or condition. For example, the pathology can be associated with a particular nucleic acid sequence or "pathological nucleic acid" (which refers to a nucleic acid sequence that contributes, in whole or in part, to pathology, for example, the pathological nucleic acid can be encoded to have a specific pathogenic or pathologically relevant mutation or The nucleic acid sequence of the polymorphic gene is related). Pathology may be associated with the expression of a pathological or pathological polypeptide that contributes, in whole or in part, to a pathology associated with a particular disease or condition. Doc . 46- 5 201130978 Off. In another embodiment, the pathology is associated, for example, with other factors such as ischemia and the like. As used herein, the term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or condition. As used herein, the phrase "parenteral administration" means a mode of administration, usually by injection, in addition to enteral and topical administration, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intralesional , heart, indoor, intracapsular, intraocular, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, sub-keratinous, intra-articular, subcapsular, subarachnoid, intraspinal, intracerebral spinal cord and intrasternal injection And infusion. As used herein, the phrase "systemic administration" and "peripheral administration" means that the target stem cell population or its differentiated progeny and/or its progeny and/or compounds and/or other substances are not directly administered to the individual. It is allowed to enter the animal system and is therefore subject to metabolism and other similar processes, such as subcutaneous or intravenous administration. The phrase "medically acceptable" is used in this context to refer to contact with humans and animal tissues without undue toxicity, irritation, allergic reactions or other problems or complications, and reasonable. The compounds/substances, compositions and/or dosage forms that are commensurate with the benefits/risk ratios. As used herein, '"pharmaceutically acceptable carrier" means a pharmaceutically acceptable substance, composition or vehicle, such as a liquid or solid, a filler, a diluent, an excipient 'solvent or encapsulating material, It involves carrying or transferring the target medicament from one organ or body part to another organ or body part. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation. As used herein, the term "drug" or "compound" refers to the individual who is involved in 152717. Doc -47· 201130978 A chemical entity or biological product, or chemical entity or combination of biological products, that treats or prevents or controls a disease or condition. Chemical entities or biological products are preferably, but not necessarily, low molecular weight compounds, but may also be larger compounds, such as nucleic acids, amino acids or carbohydrate oligomers, including (not limited to) proteins, oligonucleotides, ribose Nucleases, DNAzymes, glycoproteins, siRNAs, lipoproteins, aptamers, and modifications and combinations thereof. As used herein, the term "transplantation" refers to the introduction of a new cell (eg, a target stem cell population or a differentiated progeny thereof) or tissue (such as a differentiated cell produced from a target stem cell population) or an organ into a host (ie, a transplant recipient or transplanted individual). The term "regulation" is used consistently with its use in the art, for example to mean causing or contributing to qualitative or quantitative changes, changes or modifications of a process, path or related phenomenon. Without limitation, this change may be reduced or varied for the relative strength or activity of different components or branches of the process, path or phenomenon. A "modulator" is an agent that causes or contributes to a sputum or quantitative change, change, or modification of a process, pathway, or related phenomenon. The terms "decreasing," "decreasing," or "suppressing" are used herein generally to mean reducing a statistically significant amount. However, for the avoidance of doubt, reducing "or decreasing" or "suppressing" means reducing at least (four), such as by at least about 2%, or at least about 3%, or at least about 40%, compared to the degree of reference. , or at least about 50%, up to about 60%, or at least about 70% or at least about 80%, or at least about 9 inches. / + n 90/0 or up to and including 100% reduction (- such as 'there is no sub-presentation compared to the reference sample), or 10-100 °/ to the reference level. Any reduction between. 152717. Doc 5 •48· 201130978 The terms “increase”, “enhance” or “energy” are used throughout this document to mean increasing the static mass. • To avoid any doubt, the term “increase” or “improve” or "Activation" means an increase of at least 10% compared to the degree of reference, for example by at least about 2%, or at least about 30%, or at least about 40%, or at least about 5%, or more than the reference level, or At least about 6%, or to /' force 70/. , or at least about 8%, or at least about or at most and including any increase between 100% increase or 丨0-100%, or at least about 2 times 'or at least about 3 times' compared to the degree of reference or At least about 4 times, or at least about 5 times or at least about 10 times increased, or any increase between 2 times and 10 times or more. The term "statistically significant" or "significant" refers to statistical significance and generally means two standard deviations (2SD) below or below the normal value. This term refers to statistical evidence of differences. It is defined as the probability of rejecting the null hypothesis when the null hypothesis is actually true. The P value is often used to make a decision. As used herein, the term "substantially" means at least about 60%, or preferably at least about 70%, or at least about 8%, or at least about 90%. Up to about 95%, at least about 97%, or at least about 99% or 99% above or 70°/. The ratio of any integer between 1〇〇%. The singular forms "a", "the", "the" and "the" are used in the singular terms. Thus, for example, reference to "the method" includes one or more methods, and/or steps of the type described herein and/or it will become apparent to those skilled in the art in reading the disclosure and the like. . It should be understood that the foregoing embodiments and the following examples are illustrative only and should not be considered as 152717. Doc -49· 201130978 _ for the material of the present invention. Various changes and modifications to the disclosed embodiments may be made without departing from the spirit of this (4) and Fan Ming. Those who are familiar with the technology will be aware of all patents, patent applications and publications. The disclosure is hereby expressly incorporated by reference to the extent of the disclosure of the disclosure of the disclosure of the disclosure of the disclosure. In the case of the application for the disclosure of the disclosure of the application for the application of the singularity of the singer, the singer of the singer In the previous invention or because of any of its (4), the inventor has not authorized the disclosure in advance of this disclosure. All statements regarding the date or the contents of such documents are based on the information available to the applicant and do not constitute Any recognition of the correctness of the date or content of such documents. Washing device: One aspect of the present invention is the use of centrifugal panning to separate different target stem cell populations from peripheral blood into a subset of stem cells. Without wishing to be bound by theory, in a panning process, a peripheral blood sample is introduced into a generally funnel-shaped separation chamber located on a rotary centrifuge, and then a liquid panning buffer or a low density liquid stream is introduced into the peripheral blood sample. In the chamber. Because the flow rate of the liquid panning buffer increases as it passes through the chamber (usually in a stepwise manner), the liquid purges smaller, slower settled cells into the panning boundary of the chamber' while larger and faster settled cell migration In the chamber area to balance the centrifugal force and the sinking (drag) force. Therefore, since peripheral blood such as mobilized peripheral blood contains many different stem cell populations, one aspect of the present invention relates to the use of panning to separate such different stem cell populations present in peripheral blood into different populations, wherein the self-size is larger The stem cells fractionate smaller stem cells. 1527l7. Doc 5 201130978 In some aspects of the invention, any panning device can be used to fractionate peripheral blood samples into portions comprising different stem cell populations. In some embodiments, commercially available panning devices can be used, such as Gambro BCT, Inc. Manufactured ELUTRA® centrifuge. ELUTRA CELL SEPARATION SYSTEM® allows cell populations to be separated into multiple fractions based on size and density, allowing cell enrichment, elimination, concentration and washing to be performed in a closed functional system. ELUTRA CELL SEPARATION SYSTEM® can directly concentrate the stem cell population from leukocyte deionization products without the need for antibodies or pretreatment in less than one hour. ELUTRA CELL SEPARATION SYSTEM® uses a countercurrent centrifugal panning method in which fluid flows through the cell layer in a centrifugal field to separate the cell population. In some embodiments, other panning devices can be used including, but not limited to, COBE® Spectra separation systems, TRIMA® systems, and TRIMA ACCEL® systems (also by Gambro BTC Inc. Manufactured), as well as other commercially available panning devices used to separate blood components. In certain embodiments, a cell separator such as a COBE® Spectra separation system is used to fractionate peripheral blood. The COBE® SPECTRATM centrifuges are described in U.S. Patent Nos. 4,425,172; 4,708,712; and 6,022,306 each incorporated herein by reference. In this embodiment, the peripheral blood sample is drawn into a cell separator, such as a COBE® Spectra separation system, and the anticoagulant solution is optionally added to the blood to keep it from coagulation during the procedure. The blood/anticoagulant mixture is circulated through a centrifuge to separate peripheral blood samples into a population of stem cells, as well as monocytes from other blood components and plasma. The system will detach the isolated stem cells to 1527l7. Doc -51· 201130978 Store the collection bag and return other blood components and plasma to the patient. All tubing sets and needles used are sterile, so there is no risk of disease transmission. Other blood separation devices can be used, such as those described in the following: U.S. Patent No. 5,722,926, issued March 3, 1998, and U.S. Patent No. 5,951,877, issued on September 14, 1999; U.S. Patent No. 6,053,856 issued January 25, 2002; U.S. Patent No. 6,334,842 issued Jan. 1, 2002; U.S. Patent Application Serial No. 1/884,877, filed on Jan. 1, 2004; 848; U.S. Patent No. 6,22,326; U.S. Patent No. 6,589,526; U.S. Patent Application Serial No. 2/8/35,585; US Patent No. 2/8/18 18756 and 2009/0104626. The entire disclosures of each of these U.S. patents and patent applications are hereby incorporated by reference. Without being bound by theory, leukocyte desorption refers to a type of separation procedure that separates white blood cells from other particles and typically uses a centrifuge. The resulting collected or isolated leukocytes can then be further separated into subsets of desired cells (e. g., monocytes, lymphocytes, and granulocytes) for collection or culture as necessary, although it will be appreciated that other cells may also need to be collected. However, the collected leukocyte deposition products are often contaminated with platelets and red blood cells, which can interfere with various cell isolation and/or cell selection techniques and later culture of selected cells for therapeutic use. Therefore, it is necessary to separate white blood cells into desired subsets and to remove or reduce the number of platelets and red blood cells. Several methods have been proposed for separating or fractionating white blood cells from other particles and purifying the selected subset. One such method is a centrifugal elutriation method. In a common form of panning, cell batches are introduced into a rotating centrifuge at 152717. Doc -52- 5 201130978 Generally in the funnel-shaped separation chamber. The liquid panning buffer or low density liquid stream is then introduced into a chamber containing the cell batch. Because the flow rate of the liquid panning buffer increases as it passes through the chamber (usually in a stepwise manner), the liquid purges the smaller size of the slower settling cells into the panning boundary of the chamber, while the larger and faster settled cells migrate. In the chamber area to balance the centrifugal force and the sinking (drag) force. A relatively long panning time may be required to achieve optimal separation and purification of selected cells to relevant subsets. These long periods of time require liquid flushing buffer to continue to flow into the chamber. This in turn may require larger volumes of bags and/or multiple smaller bags to accommodate the liquid or buffer required for optimal cell separation. The use of larger volumes of panning buffer also adds additional expense to each separation method. Preferably, the panning process as used herein separates peripheral blood samples into various target stem cell populations that do not affect the function or viability of the stem cells. I is collected from peripheral blood obtained from any individual, such as a human individual, including an adult or a non-newborn child. In addition, the collection may include one or more, a set step or a collection phase. For example, one person may be subjected to at least two, at least three, or at least five collections (eg, using a separation method). In _ _ _, the total number of nucleated cells per kilogram of human body weight (four) may be one million (1 χΐ〇 6) or more than one million (eg lxl07, lxl 〇 8 ' 1X109 ' 1x10, 0 ' lxl 〇 n ' Ϊ́χΐ〇12 ^ 1X10- ix10u .  1χ10,5Ίχι〇^ιχι〇->1χ1〇^1χ10^1><1〇20)〇^^ In the preferred embodiment, the body weight and age of the donor are m φ ΐΙΛ. ^ ^ m The number of cells collected by A can be equal to or greater than lxlol 5 total nucleated cells, 152717.doc - 53- 201130978 or at least about lxl〇M, lxl〇丨3, lx1〇〗 2, 1χ1〇11, ιχι〇ι〇1χ109, lxio8' lxl〇7, lxl06, lxl〇5 total nucleated cells. Depending on the condition and quantity and quality of the stem cells collected by human donors, it is better to be at the age of "adult" or "mature" (unless used in other ways to produce different meanings in specific situations, otherwise used herein) "Adult adulthood" refers to and includes adult and non-neonatal) and/or human individual donors at a certain minimum body weight to collect stem cells. For example, when the individual is in the range of 10 to 200 kg of body weight according to one embodiment of the present invention, dry fines can be collected in any range within the range, such as 20 to 40 kg. Additionally or alternatively, 'in accordance with an embodiment of the present invention, an individual may be required to have a range of 2-80 years old (eg, 2_10, 1〇15, 12_18, 16 20^20-26 '26-30' 30-35' 30-40 '40-45 '40-5〇55-60, 60-65, 60-70 and 70-80 years old) - age. Flow Rate: In certain embodiments, the rate of liquid panning buffer flow determines the size of the target stem cells that are separated from the remaining stem cells. In certain embodiments, a target stem cell population of small stem cells (e.g., including but not limited to VSELs) can be separated from other stem cells in peripheral blood when the wash flow rate is at least about 50 ml/min. In certain embodiments, the target stem cell population of stem cells larger than the VSEL size can be separated from other stem cells in the peripheral blood when the panning flow rate is at least 7 ml/min. In certain embodiments, the target stem cell population of the stem cells can be separated from other stem cells in the peripheral blood when the panning flow rate is at least about 90 ml/min. In a certain embodiment, when the panning flow rate is at least about >90 ml/min or about 1527 l7.doc • 54 - 5 201130978 105 ml/min, the stem cell population of the stem cells can be combined with the peripheral blood. Other stem cells are isolated. In certain embodiments, a flow rate of less than about 20 ml/min can be used to collect platelets from peripheral blood. In certain embodiments, a flow rate of between about 2 ml/min to about 20 ml/min can be used to collect platelets. In certain embodiments, the first portion is collected at a flow rate of about 2 〇 ml/min or 2 〇 ml/min. In certain embodiments, between 2 〇 ml / deduction to about 50 cc / min, such as about at least 2 〇 ml / min, or at least 30 ml / min, or about 40 ml / min or about 50 ml / The second fraction is collected at a flow rate of one minute, in some embodiments, between 51 ml/min and about 70 ml/min, such as about at least 51 ml/min, or at least 60 ml/min, or about 65 ml. The third fraction was collected at a flow rate of /min or about 7 〇ml/min. In certain embodiments, between 7 liters per minute to about 90 milliliters per minute, such as about at least 71 milliliters per minute, or at least 80 milliliters per minute, or about 85 milliliters per minute or about 9 milliliters per minute. The fourth part was collected at the flow rate. In certain embodiments, between about 91 ml/min to about 105 ml/min or greater than 1 〇5 ml/min, such as about at least 91 ml/min, or at least 95 ml/min, or about 1 〇〇. Collect the fifth part at ML/min, or at a flow rate of approximately 105 ml/min. Alternatively, in certain embodiments, the fifth portion can be collected in sub-partial form, for example, the cells can be collected at one or more of the following flow rates; at least about 95 ml/min, or at least about 100 ml/min, or about 1 〇5. ML/min, or greater than 105 ml/min. This embodiment can be adapted to collect sub-portions comprising substantially different populations of target stem cells, such as _ subsections of the cells collected therein 152717.doc • 55· 201130978 is another sub-HSC containing cells and the collected cells Part contains essentially MSC. In certain embodiments, a relatively long panning time may be required to achieve optimal separation and purification of selected stem cells to a relevant subset of stem cells (e. g., a target stem cell population). These long periods of time require liquid panning (4) continuous flow in the middle chamber. This in turn may require larger volumes of bags and/or multiple smaller bags to accommodate the amount of liquid or buffer required for optimal cell separation. Again, the use of larger volumes of panning buffer adds additional expense to each separation process. In certain embodiments, as set forth in U.S. Patent No. 4,939, the disclosure of which is incorporated herein by reference in its entirety, Cell size and continuous analysis of the separated fractions with respect to other important cellular characteristics such as specific density, morphology, and cell surface markers. As used herein, the term "optimal setting" refers to a setting that results in a fractional separation: in a portion of a cell swab, there is an optimal number of cells that have specific values and cell characteristics that are within a particular range of values. For example, the optimal setting for the fractionation of blood samples around Chiang (such as mobilized peripheral blood samples) is shown in Table 1 in the rpm of the rpm (corresponding to the 825^ operation of the ELUTRA® device from the periphery of humans) The best setting for blood separation of different target stem cell populations 152717.doc •56- 201130978

Partial optimal setting (flow rate) Increase in panning yield after target pre-panning rate of target cell population (concentration factor) Part 1 > 20ml/min Platelet fraction 2 50 ml/min VSEL 0.001% 0.3% 30 Multiples 3 70 ml/min Part 4 90 ml/min WBC and RBC Part 5 > 90 ml/min HSC and MSC are as follows for separation from peripheral blood obtained from humans using the ELUTRA® device operating at a centrifuge speed of 2400 rpm Another suitable combination of flow rates for different target stem cell populations: platelets 35 ml/min VSEL 50 ml/min VSEL 70 ml/min red J&L balls, * - some HSC 90 ml/min pure HSC 100 ml/min MSCs 110 Ml/min MSCs 120 ml/min The average skilled person will be able to select the suitable flow rate and centrifugation speed for the collection of specific stem cell populations using ELUTRA® for the collection of their stem cells using a panning device other than ELUTRA®. The flow rate of the group. Using the flow rates and centrifugation speeds disclosed herein, as well as common knowledge in the art, such as the Stoke's equation mentioned below, choose to collect the appropriate flow of the desired stem cell population on another panning device. The rate will be a regular transaction. Thus, in certain embodiments, the desired target stem cell population in the fraction may be 152717.doc -57 - 201130978 less than 1% of the total number of cells in the fraction obtained by the panning separation method, for example in the case of section 2 The desired target group is VSEL (very small embryo-like cells), which is 0.3% of the total cells in component 2, but is 3 times more concentrated than the pre-panning peripheral blood sample. In certain embodiments the 'second portion (Part 2) or portion collected from a flow rate of about 50 ml/min comprises at least about 0.05% or at least about 0.1 〇/〇, or at least about 0.2%, or about 0.3%. 'Or at least about 4%, or greater than 〇.40/0 VSEL. In certain embodiments, the second portion (Part 2) or a portion of the cells collected from a flow rate of about 5 〇 ml/min can be separated into different subpopulations of cells, for example, into a substantially pure VSEL population. The permeabilized VSEL population comprising at least about 75%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% VSEL, relative to the cells comprising the total cell population. In certain embodiments, the fifth portion (portion 5) or portion from a flow rate greater than 9 〇 ml/min, such as about 105 ml/min or greater than 1 〇5 ml/min, comprises at least about 1% 'or At least about 2%, or at least about 3 〇/〇, or about 4% or at least about 5%, or greater than 5% HSC or MSC cells. In some embodiments, the fifth part (part 5) or the fraction of cells collected from a flow rate of about 5 ml/min or more than 105 ml/min can be further fractionated' so that the HSC and MSC population can be Separation from non-HSC cells and non-MSC cells' and further separation of HSC cells from MSC population. Thus, the fifth portion (Part 5) or a portion of the cells collected from a flow rate of about 90 ml/min, such as ι 5 ml/min or greater than 105 ml/min, can be separated into a subpopulation of cells 'for example, The substantially pure HSC cell population is isolated and isolated into a substantially pure MSC cell population. Relatively pure to the cells constituting the total cell population, substantially pure 152717.doc -58·5 201130978 HSC population comprises at least about 75%, preferably at least about 85%, more preferably at least about township and optimally at least about 95% HSCe is similarly The substantially pure MSC population comprises at least about 75%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% MS relative to the cells comprising the total cell population (> in the present month b) The towel covers the number of fractionation of the blood of the week 4 into sections. In other words, the d-side blood can be subjected to any number of different flow rates to produce any number of fractions. For example, the peripheral sputum can be separated into at least 4 or until eve. 5' or at least 6' or at least 7, or at least 8' or at least 9, or at least 10' or at least U' or at least 12' or greater than 12 different portions. For example, in some embodiments, 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 11, 戎$19, ·+, 〇·μ, 忒^ 12 or greater than 12 different flow rates separate the peripheral blood into a plurality of different portions. In some embodiments t, The desired yield of the target stem cell population is determined by collecting the volume as follows: Yield of the target stem cell = target of the stem cell population Collection of the target dry tissue ~~ can be as described in U.S. Patent No. 6,022,306 (which is incorporated by reference) The target flow rate is calculated as discussed in Table 1 herein. In certain embodiments, 'as in U.S. Patent No. 7,2,1, which is incorporated by reference in its entirety herein. It is revealed that cell particles with different sedimentation velocities are separated from the panning method. The sedimentation velocity (SV) of the spheroids is described as follows: 152717.doc •59· 201130978 sv = 2y2(pp-pm)g 9η r For the particle radius,

Pp is the particle density,

Pm is the density of the liquid medium, η is the viscosity of the medium, and 茗 is gravity or centrifugal acceleration. Since the particle radius is raised to a second power in the Stoke equation and the particle density does not rise', the cell size, rather than its density, greatly affects its rate of settling. This explains why larger particles generally remain in the chamber during centrifugation and smaller particles are released (if the particles have similar densities). Centrifugal panning has many limitations, as described in U.S. Patent No. 3,825,175, the disclosure of which is incorporated herein in its entirety. In most of these methods, particles must be introduced into the fluid medium stream in individual, intermittent batches to allow for adequate particle separation. Therefore, some panning methods only allow separation in the form of batches of particles and require other fluid media to transport the particles. In addition, the flow force must be precisely balanced with the centrifugal force to allow for proper particle segregation. Thus, the present invention encompasses a centrifugal elutriation process as disclosed in U.S. Patent Application Serial No. 2//756, which is incorporated herein by reference in its entirety. Peripheral blood samples for enriching a subset of stem cells by panning. In certain embodiments, a method for a method as disclosed herein is obtained from a mammalian individual (such as a human individual). The heart is the peripheral blood sample. In some embodiments 152717. Doc 5 • 60 - 201130978 'The human individual mobilizer has been previously administered to increase the yield or number of circulating stem cells in the peripheral blood. The methods of mobilization are known in the art and include, without limitation, the methods disclosed in U.S. Patent No. 6,261,549, and U.S. Patent Application Serial No. 2009/01. . In certain embodiments, the mobilizing agent administered to the individual includes, but is not limited to, G-CSF, GM-CSF, dexamethasone, CXCR4 receptor inhibitor, interleukin-1 (IL_i), mediator White _3 (il-3), interleukin _8 (IL-8), PIXY-321 (GM-CSF/IL-3 fusion protein), macrophage inflammatory protein, stem cell factor, thrombopoietin and growth A related oncogene, either in the form of a single mobilizer or in any combination of such mobilizers. Other mobilizing agents include, for example, but are not limited to, CXCR4 inhibitors, such as AMD31GG, ALX4G-4C, T22, T134, T14G, and TAK-779, which are disclosed in U.S. Patent No. 7,169,75, the disclosure of which is incorporated herein by reference. The manner of full reference is incorporated herein. In some embodiments, the peripheral gold sample is a mobilized peripheral blood sample. In certain embodiments, the individual is collected or obtained from the individual at least 2 days after administration of the mobilizer, and in some implementations, the individual is collected or obtained from the individual 4 or 4 days after the administration of the effective amount of the mobilizer. Blood Sample 0 In certain embodiments 'pre-treatment of peripheral blood samples or mobilization prior to separation of multiple target stem cell populations according to methods disclosed herein, for example, by other methods (eg, chemically lysing lymphocytes) Peripheral blood samples. Thus 'in some embodiments, for use according to this 152717. Doc-61 - 201130978 The starting material for the panning process disclosed in the method is the separation product, rather than the peripheral blood sample itself, in which the separation product is obtained from the peripheral liquid sample treatment. An individual suitable for collecting a plurality of stem cell populations from peripheral blood using methods as disclosed herein can be any individual of any age, such as any human individual. In certain embodiments, the individual is a child such as a child between about 1 and 18 years old. In some embodiments, the individual is an adult individual. In certain embodiments, the individual is a healthy individual. In certain embodiments, the individual is predisposed to a disease (eg, diabetes, cardiovascular disease, etc.), such as a mutation or genetic marker (such as a SNP, etc.) in a gene associated with an increased risk that the individual will develop a disease at a future date. Individuals. In certain embodiments, as discussed above, the individual mobilizer is administered prior to obtaining a peripheral blood sample in the individual. In certain embodiments, the individual is preferably at least 1 G, or at least _, or at least about 3 years old, preferably less than about (9) years old, or less than about 50 years old or less than about 4 years old, or less than About 3 years old or less than about 2 years old. The inventors have found that it is desirable to adjust the hematocrit range for separation. To enhance the specificity of the collected cell population, for example, in one embodiment, the hematocrit range is selected to increase the V disorder ratio. In another embodiment, the hematocrit range is selected to enhance the cryopreservation of the collected VSELs, as achieved by minimizing granulocyte collection. In another embodiment, the blood cell is selected to ensure that (4) a plurality of dry fines, such as 稽 and 朦, are collected. In one embodiment, the hematocrit range is selected to be 2-3%. In another embodiment, the hematocrit range IS is selected to be 3-4% 〇 152717. Doc •62- (|) 201130978 Use of stem cell populations enriched from peripheral jk fluids In some embodiments, the enriched target stem cell population is stored in a stem cell bank for future use, for example A disease treatment or regenerative medicine or therapy regimen is initially provided to an individual of a cell, such as the cells used in a subsequent allograft procedure. In such embodiments, the target stem cells obtained by the methods as disclosed herein are processed for long term storage in the cell bank. The advantage of this procedure is that it can store different target stem cell populations, allowing the selection of target stem cell populations that are most suitable for subsequent procedures for regenerative therapy procedures. In addition, in some cases, in tissue engineering and regenerative therapies, it may be desirable to use different ratio combinations of different target stem cell populations for different tissue engineering or regenerative therapy procedures. Thus, the present invention provides the ability to modulate the use of a target stem cell population for future use. In certain embodiments, different stem cell populations obtained from peripheral blood by methods as disclosed herein can be cryopreserved (eg, frozen) at liquid nitrogen temperature and stored for long periods of time, which can be thawed, eg, for treatment Purpose, such as regenerative therapy or medicine. In certain embodiments, portions obtained from peripheral blood can be further separated into a subset of stem cells prior to cryopreservation. In certain embodiments, different target stem cell populations obtained in each fraction can be further purified and expanded (e.g., propagated by in vitro culture) prior to cryopreservation to produce a substantially pure target stem cell population. If cold; East', the stem cell population can be stored in 1%, 50% FCS, 40% RPMI 1640 medium. Once thawed, the stem cell population can be cultured and expanded ex vivo in the presence of growth factors and/or feeder layers for a suitable period of time' subsequently implanted into the individual in need. 152717. Doc-63·201130978 The method of cryopreserving stem cells is generally known and disclosed in U.S. Patent Application Nos. 2008/0220520, 2009/0022693, 2008/0241113, and 2005/0106554, and U.S. Patent Nos. 5,759,764 and 7,112,576 and 7,604,930. This is incorporated herein by reference in its entirety. For cryopreservation, each stem cell population obtained from peripheral blood by the methods disclosed herein can be suspended in DPBS and placed on ice for at least about 15 minutes in preparation for cryopreservation. The preparation process can include adding the cryopreservation medium to the target stem cell population or to a substantially pure target stem cell population, followed by a controlled rate freezer or other suitable refrigeration system (dump-freeze cold bed system or cold) The bed container (Nalgene) subjects the mixture to several temperature reduction steps to reduce the temperature of the target stem cell population or substantially pure target stem cell population to a final temperature of about -90 °C. Suitable control rate reducers include, but are not limited to, Cryomed Thermo Form controlled rate cold beam 7554 (Thermo Electron, Corp. ), plane control rate is cold 〉 East Kryo 10/16 (TS Scientific), Gordinier, Bio-Cool--FTS system and Asymptote EF600, BIOSTOR CBS 2100 series. The cryopreservation medium can be prepared to contain the medium and DMSO. Approximately 3 ml of DPBS can be added to a container (such as a 50 ml conical tube). About 1 ml of human serum albumin (HSA) can be added to about 3 ml of DPBS and then cooled on ice for about ten minutes. About 1 ml of cooled 99% DMSO was added to HSA and DPBS to prepare a final cryopreservation medium. The cryopreservation medium and the target stem cell population or the substantially pure target stem cell population can then be placed on ice for about 15 minutes, and then the cryopreservation medium is added to the cell sample. Batch processing can be used to aliquot cryopreservation media into cell samples. Example 152717. Doc • 64· 5 201130978 For a single aliquot of approximately 100 μΐ target stem cell population or substantially pure target stem cell population can be combined with approximately 3 DPBS, 1 ml HSA and approximately 1 mi 99% DMSO. Approximately 2 aliquots of approximately 200 μΐ MSC cell suspension can be combined with approximately 6 ml DPBS, 2 ml HSA, and approximately 2 ml 99% DMSO. Approximately 5 aliquots of the cell sample can be combined with about 15 ml DPBS 'about 5 ml HSA and about 5 ml 99% DMSO. Approximately one aliquot of the cell sample can be combined with about 30 ml DPBS, about 10 ml HSA, and about 10 ml 99% D M S 。. In an alternative embodiment, other cryogenic storage media can be used. For example, cryopreservation media containing cryopreservation agents can be used to maintain high cell viability results after thawing, such as cryopreservation of embryos supplemented with Cry〇St〇r CSio or CS5 (Biolife), supplemented with propylene glycol and sucrose (vitr〇life) Medium' or SAGE medium (Cooper Surgical). Glycerin can be used with other cryopreservatives such as DMSO, or can be used alone at a concentration of about 10% in a medium containing a suitable protein. In another embodiment, the cryopreservation medium can be added to a target stem cell population or a substantially pure target stem cell population. The cryopreservation medium can be added dropwise to the target stem cell population or substantially pure target stem cell population suspended in DPBS until the total volume of the suspended hematopoietic stem cells and cryopreservation medium reaches the final desired volume of the cryopreservation cell mixture. The final cryopreserved cell medium can be transferred to a 2χ5(6) barcoded cryoquat bottle with a qc sample stored in a 5 W vial cap. Approximately 200 μl sample aliquots were removed using a pipette and aliquoted to the QC lid t. After filling the lid, there will be 4. 8 (6) sample was added to 5 152717. Doc -65- 201130978 ml in a vial. Samples should be delivered for use in infectious diseases and other analyses. The frozen vial should be placed in a CRYOMED® freezer and cooled to a temperature of at or below about _85 t by a controlled rate. The cryopreserved sample should be transferred to a storage hopper at -150 ° C or below -150 °C in liquid nitrogen vapor. Any sample that is positive for an infectious disease test should be quarantined. Any sample that is negative for the infectious disease test can be transferred to a different permanent storage. The step of lowering the temperature can be programmed in a controlled rate freezer, which first reduces the mixture of the cryopreservative and the target stem cell population or the substantially pure target stem cell population. "A target stem cell population or a substantially pure target stem cell that can be combined with a cryopreservation agent." The group is controlled to cool down to prepare for final storage in the freezer. Control rate reduction can be designed to maintain cell viability. Cryo-Med Freezer (Thermo Electron Corp. The liquid nitrogen cylinder and the portable Cryo-Med freezer can be used to control the rate reduction to prepare for the final storage in the freezer. The cells can be subjected to a controlled rate reduction in a frozen vial or freezer bag to a temperature of about -9 °C. For samples of the target stem cell population collected in the freezer bag or a substantially pure target stem cell population, the cells can be subjected to the following controlled rate reduction profile: waiting at about 4 ° C, per minute i. 〇°C to -6. 0 ° C (sample), 25 ye to ^ ^ ^ per minute to the chamber wide every minute from generation to heart (10) room per minute 1 . 〇°C to -45. 0 ° C (chamber), 1 每 per minute. 0. 〇 to _9〇 ye (chamber) and end (sample at or below _85. 0 ° C). For samples of target stem cell populations or substantially pure target stem cell populations collected in cold bundle vials, the cells can be subjected to the following controlled rate reduction profile: 152717. Doc -66 - 5 201130978 at 4. Wait at 0 ° C, l. Ot: to -3. 0 ° C (sample), per minute 10. 0°C to -20. 0 ° C (chamber), per minute 1. Hey. (: to -40. (TC (chamber), 10. per minute. 0°C to -90. 0 ° C (chamber) and end. Once the mixture of cryopreservative agent and target stem cell population or substantially pure target stem cell population is at or below about _85 °C, the cryopreservation vial is transferred to the cryogenic storage unit and is at or below about 135. (The temperature is stored in liquid nitrogen vapor, or the vial can be stored in the liquid nitrogen liquid. For example, suitable low temperature storage units include (but are not limited to) LN2 cold; East MVE 1830 (Chart Industries Cell surface markers can also be analyzed by flow cytometry to analyze fresh samples of target stem cell populations or substantially pure target stem cell populations and previously cryopreserved target stem cell populations or substantially pure target stem cell populations. Cell viability and other cellular characteristics. Samples of fresh samples of target stem cell populations or substantially pure target stem cell populations can also be analyzed after cell lysis, for example, using immunoblot analysis and similar assays. Target cell population or parenchyma for cryopreservation The upper target stem cell population can be thawed according to the thawing method described herein. In the case where the target stem cell population must be thawed or substantially pure target stem cell population, the target stem cell population or the substantially pure target stem cell population may be after thawing or in culture. Cells were analyzed by flow cytometry immediately after evaluation of a cell. The cryopreservation sample can be stirred in a 37. water bath without completely thawing the cells. The cells can be transferred to about 1 ml of cooled wash medium and mixed by inversion. The sample can be centrifuged at 2000 rpm for about seven Minutes. The supernatant can be removed and the cells resuspended in approximately 100 μL of wash medium (25% HSA, 152717. Doc • 67 · 201130978 DNAse, heparin and HBSS w/Ca++ and Mg++). In certain embodiments, the resuspended cells can be centrifuged in a Blöd Bank Serofuge for about 1 minute, the supernatant can be decanted and the cells resuspended at about 1. 2 ml of Sheath fluid and thirsty. Also within the scope of the present invention is medium to long term storage of all or part of a target stem cell population or a substantially pure target stem cell population obtained by a method as disclosed herein, such as long term storage in a cell bank. The medium- and long-term storage of the cells in the cell bank is disclosed in U.S. Patent Application Serial No. 2003/005433, the entire disclosure of which is incorporated herein by reference. At the end of the treatment, the target population of stem cells or the substantially pure target stem cell population as disclosed herein can be loaded into a delivery device (such as an injector) by any method known to those of ordinary skill in the art for placement in the recipient individual. In certain embodiments, a population of target stem cells obtained using the methods disclosed herein can be suspended in a short-term storage medium suitable for infusion into an individual, such as U.S. Patent No. 5,955,257, incorporated herein by reference in its entirety. Media disclosed in the above) In certain embodiments, a population of target stem cells that are concentrated using methods as disclosed herein can be stored (eg, for donor (eg, heterologous) or future therapeutic use of another body) Or direct transplantation (for example to promote healing after surgery). In one aspect of the invention, a plurality of stem cell populations collected by a method as disclosed herein can be further sorted into at least two subpopulations, each of which is cryopreserved or cryopreserved separately (eg, in the same In the vial). In certain embodiments, at least two stem cell subpopulations can be two stems 152717. Doc •68· 201130978 Cell subgroup. For example, portion 2 can be sorted into at least two cell subpopulations such as, but not limited to, (1) a population of stem cells or a population of VSEL enrichment and (2) a population of non-stem cells or a population eliminated by VSEL. In another example, portion 5 can be sorted into at least two subpopulations of cells, such as, but not limited to, (1) HSC stem cell population or HSC enriched population and (2) MSC stem cell population or MSC enriched Groups, and (3) non-stem cell populations or groups eliminated by HSCs or MSCs. In addition, it is envisaged that two subgroups (ie, (1) and (2) above) (and groups (3) as appropriate) may be kept at a low temperature. In some embodiments, the sorting of a portion of the stem cells to the subpopulation can be achieved by positive or negative sorting or a combination thereof. In certain embodiments, the labeling of cell surface proteins can be used to positively select or negatively select or remove stem cells. Examples of such labels are well known in the art and are shown in Table 2. Table 2: Table 2 Cell type labeled hematopoietic stem cells (HSC) C34+, CD45+, CXCR4+ endothelial progenitor cells CD34+, CD45+, CD133+, KDR+ minimal embryonic-like cells (VSEL) CD34+, CD133+, CXCR4+, SSEA4+ mesenchymal stem cells (MSC) CD34· , CD45·, CD90+, CD105+, CD106+, CD44+ In certain embodiments, the target stem cell population obtained by the methods disclosed herein or the substantially pure target stem cell population can be packaged in a suitable container in the presence of a suitable medium, as appropriate. . In certain embodiments, the package further comprises written instructions for the desired purpose, such as implanting a target stem cell population or a substantially pure target stem cell population into the individual for improvement or treatment of the disease or using 152717. Doc -69· 201130978 Method of treatment for regenerative medicine or therapy (and storage methods and/or thawing methods (if cryopreservation) as appropriate). Cell Therapy: In one embodiment of the invention 'When an individual desires such cell therapy, a population of target stem cells collected from peripheral blood of the individual by methods as disclosed herein can be introduced or transplanted back into the subject. A stem cell composition comprising a population of target stem cells collected from peripheral blood of an individual by a method as disclosed herein can be used to repair, treat or ameliorate various aesthetic or functional conditions (e.g., defects) via enhancement of damaged tissue. The target stem cell population collected from the peripheral blood of an individual by the methods disclosed herein provides an important resource for reconstructing or enhancing damaged tissue and thus represents the ability to collect a plurality of medically applicable stem cell populations from individuals of a single source. In a preferred embodiment, a population of target stem cells collected from peripheral blood of an individual by methods as disclosed herein can be used in tissue engineering and regenerative medicine to replace depletion due to developmental defects, injury, disease, or aging. And the damaged part of the body. A population of target stem cells collected from peripheral blood of an individual by a method as disclosed herein provides a different system in which a plurality of different stem cell populations can differentiate into different cells and produce a particular line of the same individual or genotype. The target stem cell population collected from the peripheral blood of the individual by the methods as disclosed herein thus provides significant advantages in individualized stem cell therapy. Additionally, such target stem cell populations and compositions thereof collected from peripheral blood of an individual by methods as disclosed herein can be used to add bulk to soft tissue regions, openings, depressions, voids in the absence of disease or trauma. Such as to "smoothing" to strengthen the soft group 152717 not related to damage. Doc 5 201130978 Weaving. The invention also encompasses multiple and continuous administration of a population of target stem cells collected from peripheral blood of an individual by methods as disclosed herein. For stem cell based treatment, the target stem cell population collected from the peripheral gray matter of the individual by methods as disclosed herein is preferably collected from an autologous or heterologous human or animal source. The autologous animal or human source is preferred. The stem cell composition is then prepared and isolated as described herein. A mononuclear cell suspension is prepared according to the present invention by introducing or transplanting a target stem cell population and/or a composition thereof collected from peripheral blood of an individual by a method as disclosed herein into a human or animal recipient. Such suspensions contain, in a physiologically acceptable carrier, excipient or diluent, the concentration of the target stem cell population collected from the peripheral a fluid of the individual by methods as disclosed herein. Alternatively, the stem cell suspension can be in a serum free sterile solution such as a cryopreservation solution. Concentrated stem cell preparations can also be used. The stem cell suspension can then be introduced into one or more sites of the donor tissue, e.g., via injection. The concentrated or concentrated stem cells can be administered in the form of a pharmaceutically or physiologically acceptable formulation or composition containing a physiologically acceptable carrier, excipient or diluent, and administered to the recipient organism. Organizations (including humans and non-human animals). Compositions containing stem cells can be prepared by resuspending the cells in a suitable liquid or solution, such as sterile saline or other physiologically acceptable injectable aqueous liquid. The amount of the components used in such compositions can generally be determined by those skilled in the art. In certain embodiments, a population of target stem cells or compositions thereof collected from peripheral blood of an individual by a method as disclosed herein can be administered by placing a stem cell suspension in an adsorbent or adherent material (eg, a collagen sponge matrix). ) 152717. On doc-71 - 201130978, insert the material containing stem cells into the relevant part or insert it into the relevant part. Alternatively, a population of target stem cells collected from peripheral blood of an individual by a method as disclosed herein can be administered by parenteral injection, including subcutaneous, intravenous, intramuscular, and intrasternal. Other modes of administration include, but are not limited to, intranasal, intrathecal, intradermal, transdermal, enteral, and sublingual. In one embodiment, the target stem cell population collected from the peripheral blood of the individual by the disclosed method can be administered by endoscopic surgery. For injectable administration, a composition comprising a population of target stem cells collected from peripheral blood of an individual by a method as disclosed herein may be suspended in a sterile solution or suspension or may be resuspended in pharmaceutically and physiologically acceptable form. The aqueous or oily vehicle to be received may contain a preservative, a stabilizer, and a material which causes the solution or suspension to be isotonic with the body fluid of the recipient (i.e., blood). Non-limiting examples of suitable excipients include water, phosphate buffered saline. 4), 〇"5 aqueous sodium solution, dextrose, glycerin, diluted ethanol and its analogs' and mixtures thereof. Illustrative stabilizers are polyethylene glycol, protein, brewing, amino acids, inorganic acids, and organic acids, which may be used singly or in the form of a mixture. The amount and route of administration are determined individually and correspond to the amount of use in a similar type of application or indication known to those skilled in the art. According to the present invention, a target stem cell population collected from peripheral blood of an individual by a method as disclosed herein can be administered to body tissues, including epithelial tissues (e.g., skin, lumen, etc.), muscle tissue (e.g., smooth muscle), blood. , the brain and various organs such as the urinary line (eg bladder, urethra, ureter, kidney, etc.). According to the general treatment method described herein, by the method disclosed herein as 1527I7. Doc -72- 5 201130978 The target stem cell population collected from the peripheral blood of an individual may comprise other cells (eg, a portion of the cell comprising the target stem cell population) and may be, for example, via intravenous (i. v) The catheter, such as any other intravenous fluid, is infused into the bloodstream of the individual to be administered to the individual. Alternatively, however, an individualized mixture of cells can be produced to provide a specific cell therapy mixture for the treatment of the individual. The integrated cell mixture, such as obtained by the separation method, can be characterized, sorted, and separated into different cell populations. Cell markers such as VSEL, MSC, and HSC markers or tissue-specific markers can be used to phenotypically characterize cell populations collected from peripheral blood. Using these markers, it is possible to separate and sort based on cell type. Therefore, the cell mixture is transformed into a cell population, which can be broadly divided into two parts: a stem cell fraction and a non-stem cell fraction. The non-stem cell fraction can be further divided into a sputum cell or a fibroblast fraction and a functional cell or a fully differentiated cell fraction. Once the peripheral blood cell mixture is sorted, the stem cells and non-stem cells can be cryopreserved and stored separately. In this way, a collection or repository of different cell populations of individuals can be generated. Alternatively, the stem cells and non-stem cell fractions can be cryopreserved together prior to use, followed by sorting and separation. In certain embodiments, 'a population of target stem cells collected from peripheral blood of an individual by methods as disclosed herein can be used to produce or differentiate into any cell type produced by the germ layer (ie, endoderm, mesoderm, and ectoderm). Groups including, but not limited to, differentiated cells, neural progenitor or differentiated cells, glial progenitor or differentiated cells, dendritic glial progenitor or differentiated cells, dermal progenitor or differentiated cells, hepatocytes or Differentiated cells, muscle progenitor cells or differentiated cells, osteoprogenitor or differentiated cells, mesenchymal stem cells or progenitor cells, pancreatic progenitor cells or differentiated cells, progenitor cells or differentiation 152717. Doc •73· 201130978 Chondrocytes, stromal progenitor cells or differentiated cells, cultured expanded stem or progenitor cells, cultured differentiated stem or progenitor cells or a combination thereof. Of particular interest are hematopoietic cells, which may include any nucleated cells' and myoblasts and fibroblasts that may be involved in red blood cell, lymphoid or myeloid mononuclear cell lines. Progenitor cells are also of interest, such as hematopoiesis, nerves, stroma, muscle (including smooth muscle), liver, lung, intestine and mesenchymal progenitor cells. Also directed are differentiated cells, such as osteoblasts, hepatocytes, granulocytes, chondrocytes, myocytes, adipocytes, neuronal cells, pancreatic cells, or combinations and mixtures thereof. In certain embodiments, a population of target stem cells collected from peripheral blood of an individual by methods as disclosed herein can be combined, recombinantly or compounded into cell therapy suitable for treating a disease in a subject and/or regenerating cells of a particular tissue. mixture. Combinations of target stem cell populations, tissue-specific progenitor cells, and optionally functional cells collected from peripheral blood of an individual by methods as disclosed herein can be used, e.g., to enhance implantation of transplanted stem cells. Thus, in one embodiment, the invention provides for the use of an autologous mixture of one or more target stem cell populations collected from peripheral blood of an individual, or a combination thereof with other functional cells, by a method as disclosed herein alone. Methods and products for implanting stem cells. In certain embodiments, the 'cell therapy product can comprise from about 10% to about 90% of a particular target stem cell population collected from peripheral blood of the individual by a method as disclosed herein, from about 丨〇% to about 8%, or From about 10% to about 60% 'or from about 1% to about 4%, or from about 10% to about 90% of the target stem cell population collected from the peripheral blood of the individual by a method as disclosed herein. In certain embodiments, the target stem cell population may also comprise a non-stem cell population such as from about 5% to about 50% functional cells, from about 5% to about 40% functional fine 152717. Doc 201130978 cells, from about 5% to about 30% functional cells, from about 5% to about 2% functional cells or from about 5% to about 1% functional cells. Thus, in certain embodiments, the isolated subject indicates that the stem cell population provides a pool of different stem cell types from a particular individual that can be maintained in culture and/or cryopreserved for future use, such as for individual use, Or selective recombination (eg, conventional mixing) for use in individualized autologous therapeutic applications in regenerative therapies. Suitable examples of such cell therapy products are automixes of HSCs and MSCs or autologous stem cell populations or other conventional blends with other functional cells of the hematopoietic system. Another example is a cell therapy product comprising an autologous mixture of PBSC 'myocardial progenitor cells and optionally cardiomyocytes. Accordingly, in another embodiment, a method of treating a patient in need thereof, comprising administering to an individual, individually or in combination (either separately or in a mixture), is collected from peripheral blood of the individual by a method as disclosed herein. An autologous mixture of one or more target stem cell populations. Stem Cell Banking In another aspect of the invention, a population of target stem cells collected from peripheral blood of an individual by a method as disclosed herein can be stored in a cell bank to support a health care insurance model ( Elective heaithcare insurance model) is a member of the group that effectively protects future diseases. Individual individuals can be selected to collect their own stem cell population from peripheral blood while they are in a healthy state, to process and preserve it for future distribution for their health care needs. Thus, 'in one embodiment, 'the target stem cell population collected from the peripheral blood of the individual by the method disclosed herein is "saved" to the stem cell bank or reservoir or storage facility, or any place where the target cell population is preserved 152717 . Doc •75· 201130978 For future use. The storage facility can be designed in a manner that keeps the target stem cell population collected from the peripheral blood of the individual safe by means of a catastrophic event such as a nuclear attack. In some embodiments, the storage facility can be underground, in a hole, or in a storage bin. In other embodiments, 'it can be on the mountainside or in outer space. The storage facility can be loaded into a masking material such as lead. According to another embodiment, a method of stem cell preservation in four steps is provided. Step A involves administering one or more mobilizers to the individual to increase the amount of stem cells in the blood surrounding the individual. Step B includes collecting at least one population of target stem cells from peripheral blood using a panning method by a method as disclosed herein wherein the individual does not have an immediate perceived health condition, for example, no individual needs to use his own target stem cell population Body transplantation to treat the condition. Step C includes preserving at least one population of target stem cells collected from peripheral blood as a deposited cell population by methods as disclosed herein. Step D includes extracting a deposited target stem cell population collected by a method as disclosed herein to autologously transplant the target stem cells into the individual. The target stem cell population is used for personalized medical diagnosis and prognostic testing. Another aspect of the present invention relates to a target stem cell population obtained from individual peripheral blood by a method as disclosed herein without using a positive selection method, which is used for personalized medical applications, such as self. Therapeutic uses, as well as individualized assays that assess the effects of human diet, pharmacogenetics, neurochemicals, and/or lifestyle on the function and viability of stem cell populations. Such assays are currently well known in the art, and Shaw includes high yield screening' where compounds, agents or environmental stimuli are contacted with stem cells and can be I52717. Doc • 76· 5 201130978 Measure the effects of functional or viability of a compound, agent or environmental stimulator on function (eg differentiation potential, proliferation, survival) and viability, and will be compared to a reference control sample (eg no compound present) , or a positive control sample, such as the presence of BMP6 or other growth factors and their analogs. In one embodiment of the invention, a target stem cell population obtained from individual peripheral blood by a method as disclosed herein can be used as an individualized assay to study and understand the individual's own, without the use of a positive selection method. Signal transduction pathway for stem cell population growth and differentiation. The use of target stem cells of an individual obtained by the methods disclosed herein is useful for assisting in the development of therapeutic applications for congenital and adult heart failure. The use of the target stem cells of the individual obtained by the methods as disclosed herein makes it possible to study the specific differentiation into different lines (e.g., osteogenesis or heart line) without the need and complexity of the time-consuming animal model. In another embodiment, a subject stem cell of an individual obtained by a method as disclosed herein can be genetically modified to carry a particular disease and/or pathological property and a particular disease or disorder phenotype. In one embodiment, the assay comprises a population of different target stem cells of a plurality of individuals obtained by a method as disclosed herein, or a differentiated progeny thereof. In one embodiment, the assay can be used to study the differentiation pathways of different target stem cell populations of an individual, such as, but not limited to, differentiation along cardiomyocyte lines, smooth muscle lines, endothelial lines, and subpopulations of such lines. In another embodiment, the assay can be used to study the pathological characteristics of different target stems, 'field populations', such as diseases and/or genetic characteristics associated with the disease or condition. In certain embodiments, the disease or condition is a monthly or cardiovascular condition or disease of a wound healing condition. In some embodiments, the individual is 152717. Doc •77- 201130978 Different target stem cell populations have been genetically engineered to contain characteristics associated with the disease or condition. Such methods of genetically engineering stem cells are well known to those skilled in the art and include the introduction of nucleic acids into cells by means of transfection, for example by, but not limited to, the use of viral vectors or other methods known in the art. In one embodiment, such target stem cells can be used to more closely adjust individual treatment. For example, by administering various drug regimens administered or presented for administration, the potential drug interaction problem can be directly determined based on the different genetic makeup of the individual and the environmental factors the individual is experiencing. In this way, the dosage regimen, dose or timing can be changed to achieve good results. In certain embodiments, MSCs are used as a tool for pharmacogenetic analysis of target stem cell populations because the target stem cells are specific to the individual's genetic makeup. For example, an individual's target stem cells can have specific variants that produce different pathological features. For example, because stem cells are derived from an individual (e. g., autologous), they may have undesirable pathological features, such as mutations and/or polymorphisms, which may contribute to the disease s or cause a poor or enhanced response to the therapeutic agent. Thus, the target stem cells obtained by the methods disclosed herein can be used to assess the response of an individual's stem cells to a particular compound, agent, and in some cases, to a therapeutic regimen (eg, effective therapeutic agent, dosage, and duration) and In some cases, the prognosis of the disease is determined. The method of performing the invention herein can be used to screen for target stem cells for agents that alleviate pathology or agents that positively affect the proliferation or function of target stem cells. In an alternative embodiment, the method of the present invention can be used with (iv) and the other 152717. Doc -78· 201130978 The stem cells of the body (eg, no mutations and/or polymorphisms) are agents that cause different responses to the individual target stem cell population (due to inherent genetic makeup or specific mutations and/or polymorphism). Such methods can be used, for example, to assess the effect of a particular drug and/or agent on an individual's stem cell population compared to other human and/or stem cell populations, thus acting as a high yield screening for personalized medicine and/or pharmacogenetics. The manner in which stem cells react to agents (especially pharmacological agents), including the timing of the reaction, is an important reflection of the physiological state of the cell. In certain embodiments, individual target stem cell populations of an individual can be easily manipulated in an experimental system that provides the advantages of target line differentiation and the ability to homogenize and manipulate the external environment. In addition, because of experimental changes in the ethical unacceptability of the human germline, the ES cell transgene pathway cannot be used in experiments involving the manipulation of human genes. The genetic traits in different target stem cell populations of human individuals allow for important applications in the field of inability to test systems in vivo due to ethical issues' and can properly generalize the human biology or disease process of a particular individual. In another embodiment, a different population of target stem cells of an individual can be used to prepare a pool of cDNA pools that are contaminated with respect to cDNA that is not preferentially expressed in cells of other lines. Individual target stem cell populations can also be used to prepare antibodies specific for the signature of cardiomyocytes and their precursors. Multiple antibodies can be prepared by injecting vertebrate cells of the invention in immunogenic form. The production of monoclonal antibodies is described in, for example, the following standard references: U.S. Patent Nos. 4,491,632; 4,472,500 and 4,444,887, and Enzymology 73B:3 (1981). Specific antibody molecules can also be made by immunization 152717. Doc •79· 201130978 A pool of robust cells or virions is brought into contact with the target antigen and produced by a pure selection of positive selections. See Marks et al., New Eng.  J.  Med.  335:730,1996 and McGuiness et al., Nature Biotechnol.  14:1449, 1996. Another alternative is to reassemble random DNA fragments into antibody coding regions as described in European Patent Application 1,094,108 A. And different target stem cell populations from the terminally differentiated individuals and other lineage cells to separate the precursor cells' antibodies can be used to identify or rescue (eg, restore phenotype) cells from the desired phenotype of the mixed cell population to achieve during the immunodiagnosis Use tissue samples for costaining purposes. In another embodiment, different target stem cell populations of the individual can be used to examine gene expression profiles during and after differentiation of different target stem cell populations in the individual. The set of genes can be compared to other subsets of stem cell populations from the same or different individuals or samples of control stem cell strains known in the art. Any suitable qualitative or quantitative method known in the art for detecting specific mRNA can be used. mRNA can be detected by, for example, hybridization with a microarray, in situ hybridization in tissue sections, by reverse transcriptase-PCR, or by Northern blotting containing poly A+ mRNA. These methods are readily used to determine the difference in molecular size or amount of mRNA transcripts between two samples. Any suitable method for detecting and comparing the amount of mRNA expression in a sample can be used in conjunction with the methods of the present invention. For example, the amount of mRNA expression in a sample can be determined by generating a pool of expressed sequence tags (ESTs) from the sample. An enumeration of the relative presentation of ESTs in the pool can be used to estimate the relative presentation of gene transcripts in the starting sample. The EST analysis of the test sample can then be performed 152717. Doc 201130978 The results are compared to the EST analysis of the reference sample to determine the relative amount of expression of the selected polynucleotide (specifically, the polynucleotide corresponding to one or more of the differentially expressed genes described herein). Alternatively, gene expression can be performed in a test sample using the Gene Expression Series Analysis (SAGE) method (Velculescu et al., Science (1995) 270:484), which includes the separation of different short sequences from specific positions within each transcript. label. Sequence tags are ligated, cloned, and sequenced. The frequency of a particular transcript within the starting sample is reflected by the number of times the relevant sequence tag encounters the sequence group. The differential display (DD) method can also be used to analyze the gene expression in the test sample. In DD, in conjunction with information about the length or location of fragments within the expressed gene, fragments defined by specific sequence delimiters (e. g., restriction enzyme sites) are used as different gene identifiers. The relative presentation of the expressed genes within the sample can then be assessed based on the relative presentation of the fragments associated with the genes within the pool of all possible fragments. Methods and compositions for performing DD are well known in the art, see, for example, U.S. Patent No. 5,776,683; and U.S. Patent No. 5,807,680. Alternatively, hybridization analysis based on the specificity of nucleotide interactions can be used to assess gene expression in a sample. Oligonucleotides or cDNAs can be used to selectively identify or capture dNAs or RNAs of specific sequence composition, and to measure the amount of rna or cDNA that hybridizes to known capture sequences in an inert or quantitative manner to provide information about the cells in the sample. Information about the relative presentation of specific silos in the pool of messages. Hybridization assays can be designed to allow for the use of, for example, array-based techniques with high density formats (including filters, slides or microchips) or by spectral analysis (e.g., mass spectrometry) based on 152717. Doc 201130978 Liquid technology to screen the relative performance of hundreds to thousands of genes in parallel. An exemplary use of an array in a diagnostic method of the invention described in greater detail below can be hybridized to an array, wherein the array can be produced according to any suitable method known in the art. A method of making a large array of oligonucleotides using light-directed synthesis techniques is described in U.S. Patent No. 5,134,854 and U.S. Patent No. 5,445,934. The heterogeneous monomer array is converted to a heterogeneous polymer array by simultaneous coupling at a number of reaction sites using a computer controlled system. Alternatively, the microarray is produced by depositing a pre-synthesized oligonucleotide on a solid substrate, as described in PCT Publication No. WO 95/355-5. Methods for collecting data from the hybridization of samples and arrays are also well known in the art. For example, a polynucleotide that produces a sample of cells can be detected using a fluorescent label, and the hybridization of the polynucleotide in the sample can be detected by scanning for the presence of a detectable label in the microarray. Methods and apparatus for detecting a fluorescently-marked object on a device are known in the art. Generally, such detection devices include a microscope and a light source that directs light at the substrate. The photon counter_ is fluorescent from the substrate, and the x-y translation stage changes the substrate position. A confocal (four) device that can be used in the subject method is described in U.S. Patent No. 5,631,734. Scanning laser microscopy is described in Shalon et al., Gen 〇me Res (Η%) 6:639. Each glory used is scanned using a suitable excitation line. The digital images produced by the scan are then combined for subsequent analysis. For any particular array element, the ratio plate of the fluorescent signal from one sample is compared to the fluorescent signal from the other sample and the relative signal is measured. In this technique, it is well known to analyze the material collected from the hybridization with (4). For example, 152717. Doc 201130978 言 “When hybridization detection includes fluorescent markers, data analysis can include the following steps: determining the fluorescence intensity as a function of substrate position from the collected data, removing outliers (ie, deviating from a predetermined statistical distribution) Data), and the relative binding affinity of the remaining data calculation targets. The resulting data can be displayed in image form. The intensity in each region varies depending on the binding affinity between the target and the probe. Pattern matching can be done manually or using a computer program. For example, U.S. Patent No. 5,800,992 describes the preparation of substrate matrices (e.g., arrays), oligonucleotides designed for use with such matrices, labeled probes, hybridization conditions, scanning hybrid matrices, and patterns generated by analysis (including comparative analysis). )Methods. In the target stem cells obtained by the method as disclosed herein, the stem cell population or stem cell population combination is contacted with the relevant agent, and by monitoring output parameters (such as marker expression, cell viability, differentiation characteristics, pluripotency, and It is similar to the parameter) to evaluate the effect of the agent. For use in assays, target stem cells can be freshly isolated using methods as disclosed herein, or in certain embodiments cultured, cryopreserved, or genetically engineered. The target stem cells can be environmentally induced variants of the pure lineage culture: for example, divided into independent cultures and grown under different conditions, such as with or without virus; presence or absence of other cytokines or combinations thereof. Alternatively, the target stem cell can be a variant having the desired pathological characteristics. For example, because target stem cells are derived from an individual (i.e., autologous), they have desirable pathological features such as mutations and/or polymorphisms that contribute to disease pathology. Thus, a pool of target stem cells obtained by a method as disclosed herein can be used to assess the response of an individual's target stem cells to a particular compound, agent, 152717. Doc -83- 201130978 and in some cases may be suitable for disease prognosis. In this embodiment, the method of the present invention can be used to screen a target stem cell population for an agent that alleviates pathology. In an alternative embodiment, the method of the invention can be used to screen for agents in which some target stem cell populations comprising specific mutations and/or polymorphisms are reacted differently than stem cells of the same type without mutation and/or polymorphism. . Thus, such methods can be used, for example, to assess the effect of a particular drug and/or agent on a target stem cell population of an individual as compared to other humans and/or cells' thus acting as a high yield screening for personalized medicine and/or pharmacogenetics. The way in which a cell reacts to an agent (especially a pharmacological agent) (including the point in time of the reaction) is an important reflection of the physiological state of the cell. The agent for the screening method may be selected from the group consisting of a chemical substance, a small molecule, a chemical entity, a nucleic acid sequence, an agent, a nucleic acid analog, or a protein or a polypeptide or a fragment thereof. In certain embodiments, the nucleic acid is dNA or RNA' and the nucleic acid analogs can be, for example, pna, pCPNA, and LNA. The nucleic acid can be single or double stranded' and can be selected from the group consisting of a nucleic acid encoding an associated protein, an oligonucleotide, a PNA, etc., such nucleic acid sequences include, for example, but not limited to, a nucleic acid sequence encoding a protein that: Transcription inhibitors, antisense molecules, ribonucleases, small inhibitory nucleic acid sequences such as, but not limited to, RNAi, shRNAi, siRNA, microRNAi (mRNAi), antisense oligonucleotides, and the like. The protein and/or peptide agent or fragment thereof can be any related protein, such as, but not limited to, a mutein, a therapeutic protein or a truncated protein' wherein the protein is typically absent or less expressed in the cell. Related proteins may be selected from the group consisting of muteins, genetically engineered proteins, peptides, synthetic peptides, recombinant proteins, chimeric proteins, antibodies, humanized eggs 152717. Doc •84· 5 201130978 Humanized antibodies, chimeric antibodies, modified proteins and their tablets. The agent can be administered to a medium in which it contacts cells (such as stem cells and ' or mesenchymal cells) and induces its effects. Alternatively, the agent may be intracellular, i.e., within a cell (such as a stem cell or a mesenchymal cell), by introducing the nucleic acid sequence into the transcript and causing it to produce nucleic acid and/or protein drug J in the cell. The agent also covers any effects and/or events on the cells. As a non-limiting example, the effect may include any effect that triggers a physiological change in the cell 'eg, but not limited to, heat shock, ionizing radiation, shock electrical pulse, light and/or wavelength exposure, uv exposure, pressure, pull ^ a large and/or reduced oxygen exposure, exposure to reactive oxygen species (R^S), ischemic conditions, fluorescent exposure, and the like. Environmental stimuli also include the intrinsic % of environmental stimuli. Exposure to a pharmaceutical agent may be continuous or intermittent. The term "agent" means any chemical substance, entity or moiety, including, without limitation, synthetic and naturally occurring non-protein f-type entities. In certain embodiments the 'related compound is a small molecule having a chemical moiety. For example, the study includes, by substitution or substitution, a substituted or substituted base, an aromatic or heterocyclic base, a sputum, a sputum, a related natural product, or the like, and/or A compound of a property, or a pool of different compounds thereof, may be selected. In certain embodiments, the agent is a related agent comprising: a known and unknown compound encompassing a poly-chemical class (mainly an organic molecule), which may include an organometallic molecule, an inorganic molecule, a gene sequence, and the like. An important aspect of the invention is the evaluation of candidate drugs, including toxicity tests and the like. Wait 152717. Doc •85- 201130978 Selective agents also include organic molecules containing functional groups necessary for structural interactions (specifically hydrogen bonding), and usually include at least one amine group, carbonyl group, hydroxyl group or carboxyl group, often including at least two functionalities Chemical group. Candidate agents often comprise a cyclic carbon or heterocyclic structure and/or an aromatic or polyaromatic structure substituted with one or more of the above functional groups. Candidate agents are also found in biomolecules, including peptides, polynucleotides, sugars, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Also included are pharmacologically active drugs, genetically active molecules, and the like as agents. Related compounds include, for example, chemotherapeutic agents, hormone or hormone antagonists, growth factors or recombinant growth factors, and fragments and variants thereof. Examples of pharmaceutical agents suitable for use in the present invention are those described in "The Pharmacological Basis of Therapeutics," Goodman and Gilman, McGraw-Hill, New York, N_Y., (1996), ninth edition, in the following sections: Water, Salts and Ions; Drugs Affecting Renal Function and Electrolyte Metabolism; Drugs Affecting Gastrointestinal Function; Chemotherapy of Microbial Diseases; Chemotherapy of Neoplastic Diseases; Drugs Acting on Blood-Forming organs; Hormones and Hormone Antagonists; Vitamins, Dermatology and Toxicology, All This is incorporated herein by reference. Also includes toxins, and biological and chemical warfare agents, see for example, Somani, S.  Μ·(ed.), “Chemical Warfare Agents,” Academic Press, New York, 1992). The agent includes all of the above classes of molecules and may further comprise a sample of unknown content. Concerned about the natural existence of natural sources (such as plants) 152717. Doc -86 - 5 201130978 Complex mixture of compounds. Although many samples will contain a solution of the compound, they can be dissolved in a sample suitable for dissolution (4). Related samples include environmental samples such as groundwater, seawater, mining waste, etc.; biological samples such as lysates prepared from crops, tissue samples, etc.; manufacturing samples, such as time courses during the preparation of medicines; and preparation of sets of compounds for analysis Human library; and similar samples. Related samples include compounds that have been evaluated for potential therapeutic value, i.e., drug candidates. The parameter is the quantifiable component of the target stem cell, specifically the component that is ideally accurately measured in a high-yield system. The parameter can be any cell nucleus or cell production 16' including cell surface determinants, receptors, proteins or their conformation or post-translation (4), lipids, carbohydrates, organic or inorganic molecules, nucleic acids (eg mRNA, DNA, etc.) or Source, part of the cellular component, or a combination thereof. Although most parameters will provide a quantitative readout, semi-quantitative or qualitative results will be acceptable in some cases. Readouts may include a single assay value' or may include an average, median or variance, and the like. Characteristically, for each parameter from a large number of identical assays, the range of parameter readout values will be obtained. The range of values for each set of test parameters is obtained by predicting variability and using the standard method and the heart to provide a single-value common statistical method. Compounds including candidate agents are obtained from a variety of sources, including libraries of synthetic or natural compounds. For example, numerous methods can be used to randomly and guide the synthesis of a variety of organic compounds, including biomolecules, including the performance of randomized oligonucleotides and oligopeptides. Alternatively, a collection of natural compounds in the form of bacterial, fungal, plant and animal extracts is available or readily produced. In addition, collections and compounds produced naturally or synthetically are via the well-known chemistry, physics and 152717. Doc *87- 201130978 Biochemical methods are easily modified and can be used to generate combinatorial collection libraries. Known pharmacological agents can be directed or randomly chemically modified: for example, deuterated, alkylated, brewed, guanylated, etc. to produce structural analogs. Typically, the agent is combined with a drug-free cell, and the agent is screened for the effect on the stem cell population by adding the agent to at least one = usually a plurality of stem cell samples. The change in parameters of the reaction is measured and the results are evaluated by comparison to a reference culture (e.g., in the presence and absence of a medicament, obtained with other agents). ’. In certain embodiments, the agent is preferably added to the culture medium of the stem cells in the culture in a solution or in a readily soluble form. The agent may be added to the additional static solution either in a discontinuous or continuous flow, or alternatively, by addition of a compound bolus, either separately or incrementally, in a flow-through system (flQW_through system). In a flow-through system, two fluids are used, one of which is a physiologically neutral solution' and the other is the same as the added test compound: Solution: passes the first fluid over the stem cells, then passes through the second fluid . In a single solution method, a test compound boluse is added to the volume of the culture medium surrounding the cells. The overall concentration of the media components should not vary significantly depending on the addition of the bolus or between the two solutions in the flow-through process. In certain embodiments, the formulation of the agent does not include other components, such as preservatives, which can have a significant effect on the overall formulation. Accordingly, preferred formulations consist essentially of a biologically active compound and a physiologically acceptable carrier (e.g., water, ethanol, DMS0, etc.). However, if the compound is a solvent-free liquid, the formulation may consist essentially of the compound itself. A plurality of tests can be operated in parallel with different drug concentrations to obtain various concentrations

152717.doc •88· CD 201130978 degree difference response. As is known in the art, determining the effective concentration of a pharmaceutical agent typically employs a concentration range resulting from a 1:1 G or other logarithmic unit dilution. The column is diluted to further refine. Usually, if necessary, the concentration can be used as the negative control, that is, at zero concentration or below the drug's debt level or at or below the concentration of the agent that does not have a phenotypic change. Depending on the situation, the stem cell population selected (iv) can be manipulated to express the desired genetic product. Gene therapy can be used to modify cells to replace gene products or to add or reject gene products. In certain embodiments, genetic engineering is performed to facilitate tissue regeneration, treatment of Qianxinliang cell survival (e.g., prevention of rejection) after implantation into an individual towel. Alternatively, in an embodiment the intermediate leaf cell line is genetically engineered and transfected prior to use as a trophoblast for stem cells, or alternatively, mesenchymal cells can be transfected while serving as a trophoblast for stem cells. Techniques for transfecting cells are known in the art. Those skilled in the art can envision the transfer of beneficial properties to transfected mesenchymal cells or, if stem cells are used for transplantation (discussed in more detail below), more indirectly to recipient stem cells and/or individual genes. . Depending on the embodiment, the added gene may ultimately remain in the recipient cell and all of its progeny t, or may be retained only temporarily. For example, a gene encoding an angiogenic factor can be transfected into a sputum cell isolated from smooth muscle. These genes will be useful for inducing collateral vessel formation when smooth muscle tissue is regenerated. In some cases, it may be desirable to transfect cells with more than one gene. In some cases, a gene product needs to be secreted. In such cases, the gene product preferably contains a secretory signal sequence that facilitates secretion of the protein. For example, if the desired gene product is an angiogenic protein, those skilled in the art can use conventional gene manipulation (see, for example, Nabel et al., 1993) to select a sequence having a native 彳s number. An angiogenic protein, such as vegf, or a modified gene product to contain this sequence. The desired gene can be transfected into cells using a variety of techniques. Preferably, the expression vector is used to transfect the gene into the cell. Suitable expression vectors include plastid vectors (such as those available from Stratagene, Madison Wis.), viral vectors (such as replication defective retroviral vectors, herpes viruses). , adenovirus, adeno-associated virus and lentivirus) and non-viral vectors (such as liposomes or receptor ligands). EXAMPLES Reference will be made in detail to the embodiments of the invention, examples of which are illustrated in the accompanying drawings. In certain embodiments, the invention includes an ELUTRA® centrifuge manufactured by Gambbr® BCT, Inc., Lakewood, Colorado. Although the embodiments of the present invention are described in combination with an ELUTRA® centrifuge, this reference is for illustrative purposes only and is not intended to limit the invention in any way. It will be appreciated that other centrifuges may be used in embodiments of the invention including, but not limited to, COBE® Spectra separation systems, TRIMA® systems, and TRIMA ACCEL® systems (also manufactured by Gambro BTC Inc.), as well as for separating blood. Other panning devices for the components. Example 1 The detached mobilized PB cells were panned to separate/enrich MSCs. I52717.doc 201130978 28 studies were performed after two consecutive doses of G-CSF injection (480 μg/day, subcutaneous) and 3-4 hours after total nucleated cells (TNC) were collected by separation in the third chamber. A group of donors. Size-based MSC enrichment was performed by panning: freshly isolated cells were solubilized in lxBDPharm lysis buffer at a ratio of about 1:10 (volume/volume) to remove red blood cells. After washing, the cells were counted and 2-2.5 x 101 () total nucleated cells were loaded onto an ELUTRA® Cell Separation System (Caridian BCT) at a concentration of 1 x 10 8 cells/ml. The cells were then collected at different flow rates in 900 ml PBS + 0.5% HS A medium in each bag. Six fractions are typically collected at a centrifuge rate of 2400 rpm. Finally, all of the cells were transferred to tubes and briefly centrifuged at 600 xg for 15 minutes. The size of the part was confirmed by evaluating the SSC and FSC (Fig. 3). CFU-F assay: The possible MSC enrichment of the ELUTRA® fraction was tested by plating the cells in the CFU-F assay. Two million cells/parts were applied to 100 mm tissue culture dishes and supplemented with 10 mL DMEM + 10% FBS medium. The culture was maintained in a 37 °C incubator containing 5% C02 for two weeks without interference. Two weeks later, the culture was fixed with methanol and stained with Giemsa. Adhesion community characteristics of MSCs formed from sections 4 and 5 (Fig. 4) 染色 Staining of fractionated isolates: Flow cytometric analysis was used to determine whether certain ELUTRA fractions were enriched in MSCs. Samples from the ELUTRA® section were stained using two sets of antibodies: Group 1: CD45, CD105, CD90, and Group 2: CD271. Staining was carried out on ice in PBS containing 0.5% human albumin for 30 minutes. The cells were washed and analyzed by FACS in the form of CD457CD105+, CD457CD90+ and CD457CD271+ by 152717.doc-91 · 201130978. The cells characterized by CD45·/CD105+, CD457CD90+ and CD271+ were partially enriched in parts 4 and 5 compared to the starting group of the isolated product (Fig. 5). G-CSF mobilizes the MSC into the peripheral blood. Figure 6 shows an increase in the proportion of MSCs after mobilization and in the resulting precipitated product. Herein, MSCs are identified by detecting cells expressing CD457CD317CD105+/CD73+ or CD457CD347CD90+/CD105+/CD29+. REFERENCES All references, patents, and patent applications, which are hereby incorporated by reference in their entirety herein in their entireties in the entireties in BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Figure 1 shows a graph showing that very small embryonic stem cells (VSELs) can be enriched by size-based separation (panning) of various cell fractions in the separation product. Part 1 (flow rate 20 mL/min), part 2 (50 mL/min), part 3 (70 mL/min), 咅p 4 (90 mL/min), 咅p 5 (>90 mL/ Min). Part 2 is highly enriched in VSEL and can be used to collect purified VSEL populations for clinical use. Fig. 2. Fig. 2 is a photograph showing the separation products from peripheral blood divided into five individual parts by size. Figures 3A-F show self-eutation of human peripheral blood enriched MSCs. The ELUTRA® cell separation system was used to separate G-CSF from mobilized and isolated blood. The MSCs were enriched in sections 4 and 5. Figure 3A - Separation of blood. Figure 3B - Part 1. Figure 3C - Part 2. Figure 3D - Part 3. Figure 3E - Parts 4 and 5. Figure 3F - Part 6. Figures 4A-E show cells of the panned portion that were plated in the CFU-F assay. Adhesion cell populations are produced from parts 4 and 5. Figure 4A - Part 2. Figure 4B - Part 3. 152717.doc -92· 201130978 Figure 4C - Parts 4 and 5. Figure 4D - Higher magnification of parts 4 and 5. Figure 4E - Higher magnification of parts 4 and 5. Figures 5A-B show that the MSC is mobilized into the peripheral blood. The patient received two consecutive injections of G-CSF subcutaneously. On the 0th day before the G-CSF injection, the degree of mobilization of mesenchymal stem cells was monitored on the 3rd day after the second G-CSF injection and in the isolated product. Figure 5A: Assessment of MSC mobilization by assessment of CD45VCD317CD105+/CD73+ cells. Figure 5B: Assessment of MSC mobilization by detection of CD457CD347 CD90+/CD105+/CD29+ cells. Analyze a set of "n=28" donor samples. Figures 6A-B show FACS analysis of MSC phenotypes in the ELUTRA® section. Figure 6A: CD45_/CD105+ cells in sections 4 and 5 were enriched compared to the other fractions. Figure 6B: Parts 4 and 5 show an increase in the proportion of CD457CD105+, CD457CD90+ and CD271+ cells compared to the starting group of the isolated product 0 152717.doc -93-

Claims (1)

201130978 VII. Patent application scope: 1. A method for separating a target cell population from peripheral blood, comprising: (a) flowing a peripheral blood sample from an individual through a fluid chamber of a panning device at a first flow rate, wherein The first flow rate such that cells in the peripheral blood sample that are smaller than the target cell population flow through the fluid chamber and trap the target cell population in the fluid chamber; (b) the first flow rate Raising to a second flow rate to cause the target cell population to flow through the fluid chamber; and 0) collecting the target cell population as the target cell population flows through the fluid chamber; wherein steps (a), (b) And (c) does not include the use of positive selection techniques. 2. The method of claim 1, wherein (1) the first flow rate is selected such that cells of the peripheral blood sample that are smaller than minimal embryonic stem cells (VSELs) flow through the fluid chamber and trap VSELs in the fluid chamber In the chamber, and (1) selecting the second flow rate to cause the VSEL to flow through the fluid chamber. 3. The method of claim 2, wherein (1) the first flow rate is about 35 ml/min and the second The flow rate is about 5 〇 7 〇 ml / min. 4. The method of claim 1, wherein (1) the first flow rate is selected such that cells of the peripheral blood sample that are smaller than the mesenchymal stem cells (MSC) flow through the • a fluid chamber and trapping the MSC in the fluid chamber ' and (Π) selecting the second flow rate to cause Msc to flow through the fluid chamber. 5. The method of claim 4 wherein (1) the first The flow rate is about 1 〇〇 ml/min and the second flow rate is about i 1 〇 12 〇 mi / min. 6. The method of claim 1, wherein (1) the first flow rate is selected such that 152717. Doc 201130978 The cells in the peripheral blood sample that are smaller than hematopoietic stem cells (HSC) flow through the body cavity to Hsc is trapped in the fluid chamber, and (n) the second flow rate is selected to cause the HSC to flow through the fluid chamber. The method of claim 6, wherein (1) the first flow rate is about 9 〇 ml The second flow rate is about 100 ml/min. The method of any one of clauses 1 to 7, wherein the positive selection technique is an immunoselection method or an immunodense selection method. The blood sample is a method in which the peripheral blood sample is 9. The method of any one of claims 1 to 8 is a human peripheral blood sample. 10. The method of any one of claims 1 to 9 mobilizes a peripheral liquid sample. 11. The method of claim 10, wherein the peripheral blood sample is obtained from an individual who has been administered the mobilizer for 4 or less days. 12 · If the method of any one of items 1 to 11 is specified, Dan A. The collected target cell population is cryopreserved. 13. A method according to any one of claims 1 to 12, wherein the target cell population collected is administered to the individual. If you request the method of item 3, the target cell group collected by M m should be the most The method of providing the peripheral blood. The method of claim 13 or claim 14, wherein the collected target cell population has been expanded in vitro prior to the sputum and the individual. 16. The method of claim 15 Wherein the individual is a human individual. 17. The method of any one of claims 13 to 15 wherein the cell population is VSEL. wherein the remotely collected target 152717d〇C -2- 5 201130978 18. as claimed in claims 13 to 15 A method wherein the target cell population collected is MSC. The method of any one of claims 13 to 15, wherein the collected target cell population is HSC. 20. A method of isolating a target cell population from peripheral blood, comprising: - (a) flowing a peripheral blood sample from the individual through a fluid chamber of the panning apparatus at a first flow rate, wherein the first flow rate is selected So that the first target cell population flows through the fluid chamber and is collected to trap the first residue of the peripheral blood sample in the fluid chamber; and (b) increase the first flow rate to the first a second flow rate such that a second target population of cells flows through the fluid chamber and is collected, and the second remainder of the peripheral blood sample is retained in the fluid chamber; wherein steps (a) and (b) Does not include the use of positive selection techniques. 21. The method of claim 20, further comprising: (c) increasing the second flow rate to a third flow rate to cause a third target population of cells to flow through the fluid chamber and collecting A third residue of the peripheral blood sample is carried in the fluid chamber; wherein step (C) does not involve the use of a forward selection technique. 22. The method of claim 21, further comprising: (d) increasing the third flow rate to a fourth flow rate such that a fourth target population of cells flows through the fluid chamber and is collected, and the periphery is A fourth residue of the blood sample is worn in the fluid chamber; wherein step (d) does not involve the use of a positive selection technique. 23. The method of claim 22, further comprising: 152717.doc 201130978 (e) increasing the fourth flow rate to a fifth flow rate such that a fifth target population of cells flows through the fluid chamber and is collected, The fifth remainder of the peripheral blood sample is trapped in the fluid chamber; wherein step (e) does not involve the use of a positive selection technique. 24. The method of claim 23, further comprising: (f) increasing the fifth flow rate to a sixth flow rate such that the sixth target population of cells flows through the fluid chamber and is collected, and the periphery is The sixth residue of the blood sample is trapped in the fluid chamber; wherein step (f) does not involve the use of a forward selection technique. 25. The method of claim 24, further comprising: (g) increasing the sixth flow rate to a seventh flow rate such that the seventh target population of cells flows through the fluid chamber and is collected A seventh residue of the peripheral blood sample is trapped in the fluid chamber; wherein step (g) does not involve the use of a positive selection technique. 26. The method of claim 20, wherein (1) selecting the first flow rate such that the first target population of cells is less than cells of minimal embryonic stem cells (VSELs), and (ii) selecting the second flow rate to The second target cell population is made VSEL. 27. The method of claim 26, wherein (1) the first flow rate is about 35 ml/min and the second flow rate is about 5 〇 7 〇 mi/min. 28. The method of claim 22, wherein (1) selecting the third flow rate such that the first target population of cells is less than a hematopoietic stem cell (HSC) cell, and (Π) selecting the fourth flow rate 'to enable the first The second target cell population is HSC. The method of claim 28, wherein (1) the third flow rate is about 9 〇 ml/min and the fourth flow rate is about 100 ml/min » such as s The method of claim 23, wherein (1) selecting the fourth flow rate such that the first target cell population is smaller than the mesenchymal stem cells (MSC) cells, and (1) selecting the fifth flow rate to enable the The second target cell population is MSC. The method of claim 30, wherein (1) the third flow rate is about 1 〇〇 ml/min and the fourth flow rate is about 110-120 ml/min. The method, wherein: (I) the first flow rate is about 35 ml/min and the first target cell population is platelets; (II) the second flow rate is about 5 〇ml/min and the second target cell population Is VSEL; (iii) s second second flow rate is about 7 〇 mi / min and the third target cell population is VSEL; (IV) the fourth flow rate is about 9 〇 ml / min and the fourth target cell The group is red blood cells, (v) the fifth flow rate is about 1〇〇ml/min and the fifth target cell population is HSC; (vi) the sixth flow rate of shai About 11 〇ml/min and the sixth target cell population is MSC; and (vii) the seventh flow rate is about 12 〇ml/min and the seventh target cell population is MSC. As claimed in claims 20 to 32 A method, wherein the positive selection technique 152717.doc 201130978 is an immunoselection method or an immunodense selection method, wherein the peripheral blood sample wherein the peripheral blood sample liquid sample is obtained from a administered one of which is collected. The method of any one of claims 20 to 33 is a human peripheral blood sample. The method of any one of claims 20 to 34, wherein the method of claim 35, wherein The peripheral blood mobilizer is 4 曰 or less than 4 days. 37. The method of claim 20, wherein the standard cell population is cryopreserved. 38. The method of any one of claims 2G to 37 Wherein at least one of the target cell populations collected is administered to the individual. 39. The method of claim 38, wherein at least one of the four target cell populations collected in the scorpion beta Invest in individuals who originally provided the peripheral blood. The method of 4, 38 or 39, wherein the collected target cell populations > one has been expanded in vitro prior to administration of the individual. 41. As claimed in any of claims 38 to 40. Method, 1 body. Bazhong 3 hai individual is a human being, wherein the collected items are the items collected by the objects 42. At least one of the method target cell groups of any one of claims 38 to 41 is VSEL. 43. The method of any one of claims 38 to 41, wherein at least one of the target cell populations is Msc. 44. The method of any one of clauses 38 to 41, wherein at least one of the target cell populations is an HSC. The purpose of the collection is 45. The species from the peripheral blood separation target cell group 7 contains: 152717.doc 201130978 (a) The peripheral blood sample from the individual flows through the fluid chamber of the panning device at the first flow rate ' wherein the first flow rate is selected such that the first target population of cells flows through the fluid chamber and is collected, and the first residue of the peripheral blood sample is trapped in the fluid chamber; (b) The first flow rate is advanced to a second flow rate such that the second target population of cells flows through the fluid chamber and is collected, and the second remainder of the peripheral blood sample is trapped in the fluid chamber; c) Repeat step (b) as appropriate until the desired number of target cell populations are collected; where steps (a), (b) and (c) do not include the use of positive selection techniques. The method of any of claims 2, 3, 17, 26, 27 or 42, wherein the peripheral blood sample is a human peripheral blood sample having a hematocrit ranging from about 2-3%. 47. A method of reducing granulocytes in a composition of an individual peripheral blood VSEL comprising 'separating VSEL from other somatic cells by a separation procedure, wherein the isolated cells have a hematocrit of 2-3%. 48. A method of increasing the yield of a VSEL obtained in a downstream treatment of a separation product' by selecting a hematocrit range of 2-3%, thereby reducing the granulocyte content in the collected separation product. 49. A VSEL product' produced by the method of any one of claims 2, 3, 26 or 27. An MSC product is produced by the method of any one of claims 4, 5, 30 or 31. 51. An HSC product produced by the method of any one of claims 6, 7, 28 or 29. 152717.doc