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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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  • C12N5/069—Vascular Endothelial cells
  • C12N5/0692—Stem cells; Progenitor cells; Precursor cells
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  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/10—Growth factors
  • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/10—Growth factors
  • C12N2501/145—Thrombopoietin [TPO]
• • C—CHEMISTRY; METALLURGY
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/20—Cytokines; Chemokines
  • C12N2501/26—Flt-3 ligand (CD135L, flk-2 ligand)

Definitions

• the invention relates to cell populations and methods of producing them.
• the invention provides enriched stem cell containing populations that can be expanded, and thus can be of great value to patients in need of cellular therapy, e.g., cancer therapy, immunotherapy, and gene therapy.
• Preferred populations include at least 5% human non-fetal uncommitted hemangioblasts, i.e., common precursors of hematopoietic cells and endothelial cells; by "uncommitted” it is meant that the hemangioblasts are not yet committed to either lineage, i.e., under the proper conditions the cells can become either hematopoietic cells or endothelial cells.
• hemangioblasts are stable, not transient, and are present in the tissue of fully developed individuals, such as in newborn infants and adults. We have, in fact, discovered that these hemangioblasts can be isolated from cord blood following birth. The presence of hemangioblasts in non- embroid tissue was unexpected and presents novel opportunities..
• the uncommitted hemangioblasts can be stimulated to become hematopoietic cells or endothelial cells, by selecting appropriate growth factors in which to expand the population, as will be discussed in detail below.
• these hemangioblasts can be amplified (i.e., the number of hemangioblasts can be increased) , and/or they can be differentiated to provide a supply of hematopoietic cells, for example to patients who are immune compromised or require gene therapy with hematopoietic cells.
• the hemangioblasts When supplied with a different cocktail, the hemangioblasts can be amplified and/or can be differentiated to become endothelial cells, useful for example in wound healing, e.g., healing of slow or non- healing diabetic sores.
• the endothelial cells can also be transfected ex vivo, e.g., with genes which produce angiogenic factors, and used in gene therapy, for example to stimulate angiogenesis in patients with vascular or cardiac insufficiency. Recent studies have demonstrated the feasibility of cytokine gene transfer to enhance the antitumor activities of host immune cells. Endothelial cells forming the vascular supply of tumors may be useful vehicles for the delivery of cytokine molecules in order to effect tumor immunotherapy. Ojeifo, et al . , Cytokines Mol Ther 1996 Jun;2 (2) : 89-101.
• the invention features a method for providing a cell population containing non- fetal hemangioblasts.
• the method includes (a) providing a first cell population containing non-fetal hemangioblasts; and (b) growing the first cell population under conditions that promote the proliferation of the non-fetal hemangioblasts.
• the invention also features cell populations formed by expansion of a population containing non-fetal hemangioblasts.
• the conditions are such that the number of said non-fetal hemangioblasts and their proximity to each other are sufficient to increase the proportion of non-fetal hemangioblasts in the population.
• the method includes, prior to the growing step, enriching the first cell population for non-fetal hemangioblasts.
• the method also includes separating the non- fetal hemangioblasts from other cells in the cell culture, e.g., by a negative selection process.
• the separating step is performed concurrently with, intermittently during, or following, the growing step.
• the separating step is performed more than once during cell proliferation, e.g., every 5 to 10 days.
• the growing step includes providing at least one growth factor, more preferably a cocktail of growth factors, to the cell population during cell proliferation.
• At least some of the non-fetal hemangioblasts preferably at least 2%, more preferably at least 5%, more preferably at least 15% and most preferably at least 25%, are uncommitted human hemangioblasts.
• At least some of the non-fetal hemangioblasts preferably at least 2%, more preferably at least 5%, more preferably at least 15% and most preferably at least 25%, are CD 34-, Lin- cells.
• the percentage of cells that are CD 34-, Lin- and/or are uncommitted human hemangioblasts is higher in the enriched cell culture than in the starting cell culture.
• the uncommitted human hemangioblasts are characterized as: CD2 " , CD3 ⁇ , CD14 " ,
• the invention features an enriched cell population comprising non-fetal hemangioblasts, the enriched cell population resulting from expansion of a starting cell population containing fewer non- fetal hemangioblasts than the enriched cell population.
• the starting cell population contains at least 10% fewer non-fetal hemangioblasts than the enriched cell population.
• the percentage of cells that are non- fetal hemangioblasts in the enriched cell culture is the same as or higher than the percentage of cells that are non-fetal hemangioblasts in the starting cell culture.
• the invention also features a composition of cells in which at least 2%, more preferably at least 5%, more preferably at least 15%, and most preferably at least 25% of the cells are non- fetal hemangioblasts, and methods of making such a composition.
• the non- fetal hemangioblasts are human uncommitted hemangioblasts that are Lin “ cells and are characterized as: CD2 " , CD3 " , CD14 “ , CD16 “ , CD19 “ , CD24 “ , CD56 “ , CD66b “ , glycophorin A " , flk- 1 + , CD45 + , CXCR4+, MDR+ .
• Fig. 1 is a schematic diagram illustrating that hemangioblasts are the common precursor of hematopoietic and endothelial cells.
• Fig. 2 is a highly enlarged diagrammatic view of a system for positive selection of a target cell (a hemangioblast) .
• Fig. 2a is a highly enlarged diagrammatic view of a system for negative selection of a non-target cell (a non-hemangioblast) .
• Figs. 3 -3d are schematic diagrams of alternative modes of operation of systems according to different embodiments of the invention.
• Fig. 4 is a schematic flowchart showing a method, according to one embodiment of the invention, of treating a patient with hematopoietic stem cells.
• Fig. 5 is a graph showing the growth kinetics of CD34+/CD38-/Lin- and CD34-/CD38-/Lin- target cells over a 30 day culture period.
• Fig. 6 is a group of dot plots showing the population profile of reselected cells prior to subsequent reselection using CD34 vs. Lineage (Lin) markers over a three week period.
• Fig. 7 is a dot plot showing the population profile of initial negatively selected target populations used to inoculated cultures.
• Fig. 8 is a group of dot plots showing the population profile of reselected cells at week three of culture using CD45 vs. MHC I and MHC II and CD34 vs. Flk- 1.
• Fig. 9 is a group of dot plots showing the population profile of reselected cells at week three of selective culture using CXCR4 vs. Flk-1 and Pgp (MDR) vs. Flk-1.
• the invention broadly features a method of enriching the precursor cell content of a population containing non- fetal hemangioblasts (hereafter referred to as "target cells”) .
• target cells non- fetal hemangioblasts
• the cells are CD 34-, Lin- cells or uncommitted hemangioblasts that are characterized as: CD2 " , CD3 " , CD14 “ , CD16 “ , CD19 “ , CD24 “ , CD56 “ , CD66b “ , glycophorin A “ , flk-l ⁇ CD45 + , CXCR4 + , MDR + .
• Selection Preferred methods of expanding and enriching the population include selecting target cells from non-target cells in the cell population, concurrently with proliferation, intermittently during proliferation or following proliferation.
• Cell proliferation and cell selection can be carried out using an almost infinite variety of different techniques and settings, of which only a few are described below by way of example. Many other techniques will be readily perceived by those skilled in the art, for example selection by flow cytometry, and selection by using chemical agents to kill unwanted cells.
• the preferred selection methods used in the invention can broadly be classed as positive selection (providing a selection element having an affinity for, i.e., "selecting", target cells) and negative selection (providing a selection element having an affinity for, i.e., "selecting", non-target cells). These two selection techniques, used alone or in combination, allow unwanted cells to be removed from the system and target cells to be harvested whenever desired.
• FIG. 2 An example of a positive selection technique is illustrated diagrammatically in FIG. 2. Briefly, one or more anti-dextran conjugated antibodies specific for the predetermined target population is introduced into the culture. After a specified incubation time, a magnetic dextran iron particle colloid is introduced into the cell suspension. A Cell/Antigen/Antibody/Anti- dextran/Dextran/1ron Complex forms. This complex is then passed through a magnetic field. Positively selected cells remain in the magnetic field while cells which do not have the iron conjugated complex are removed. After capture and rinsing the magnetic field is removed and the positively selected predetermined target population is returned to the nutrient medium.
• FIG. 2A An example of a negative selection technique is illustrated diagrammatically in FIG. 2A. Briefly, one or more anti-dextran conjugated antibodies specific for a predetermined population which is not of the predetermined target population is introduced into the culture. After a specified incubation time, a magnetic dextran iron particle colloid is introduced into the cell suspension. A Cel1/Antigen/Antibody/Anti- dextran/Dextran/lron Complex forms. This complex is then passed through a magnetic field, removing cells not of the predetermined target population from the nutrient medium. The predetermined target population is collected downstream and returned to the nutrient medium. Clearly, many other techniques can be utilized for both positive and negative selection, as long as the desired affinity is provided by the selection element.
• the selection element can include other components in addition to the antibody molecules that are used to perform the selection (the "selection molecules"), e.g., a solid support to which the selection molecule is bound.
• the solid support can be formed of a material that will aid in performing the selection or in maintaining the selection molecules in a desired position or introducing and removing them from the system.
• the selection molecule can be bound to iron or other magnetic particles to allow the selected cells to be easily removed from the system by application of a magnetic field and then collected by removal of the magnetic field.
• the selection molecules can be bound onto the wall of a vessel containing the nutrient medium, or of a chamber through which the nutrient medium flows during the method.
• Glass or other inert, impermeable beads can also be used as a solid support. If beads or other particles are used, they can be provided in a packed configuration, through which the nutrient medium flows, or can be introduced into the system in a loose form, suspension, or in any desired type of array. As will be readily understood, a wide variety of other solid supports can be used.
• the selection element can be used in a variety of modes of operation in which nutrient media is supplied to and removed from the system in different manners. These modes of operation range from a selective batch culture (FIG. 3) , in which nutrient media is supplied at the beginning of cell proliferation and is neither added to nor removed, to continuous flow or recycled flow cultures (FIGS. 3C and 3D, respectively) in which either fresh or recycled nutrient media flows through the system substantially continuously. These alternative modes will be discussed in detail below.
• a nutrient medium is introduced into a vessel, and a starting sample of cells is also introduced into the vessel.
• nutrient medium may or may not be exchanged.
• selected cells are physically selected, i.e., separated from other cells in the nutrient medium by binding to a selection element, either continuously, intermittently or following cell proliferation.
• These selected cells may be cells of a target population, if positive selection is used, or unwanted cells, if negative selection is used. Dual (positive and negative) selection can be accomplished by providing positive selection molecules on the surface of the vessel, beads, baffles, impellers, etc. while removing unwanted cells by negative selection.
• cells may be positively or negatively selected outside of the culture vessel and then returned.
• the selective semi -batch (3A) and selective fed batch (3B) modes of operation are similar to the selective batch mode with regard to positive and negative selection. The significant difference between these three modes is in the treatment of the nutrient medium.
• the semi-batch mode allows for a partial refreshment of spent medium with new medium and the fed batch mode allows for an incremental increase in the medium volume over time.
• Cell growth and selection can also be performed in a continuous (FIG. 3C) or recycling (FIG. 3D) mode of operation.
• the system includes a chamber having an inlet and an outlet, and nutrient media is caused to flow through the chamber from the inlet to the outlet.
• continuous mode new nutrient media flows through the chamber from a source or reservoir, while in recycling mode the same nutrient media is cycled through the chamber repeatedly.
• a system can be configured to be run alternatively in either continuous or recycling mode. Any desired selection element can be used in these modes of operation.
• a number of parameters can be varied to affect the rate and purity of the cell output obtained during cell proliferation.
• the concentration, type and combination of growth factors can be varied to acheive a desired result.
• Suitable growth factors for promoting cell proliferation include Stem Cell Factor (SCF; R&D Systems Catalog No. 255-SC-010) , Thrombopoietin (TPo; R&D Systems Catalog No. 288-TP-005) and FLT3 (R&D Systems Catalog Nos. 308-FK-005 and 308-FK-025) , all of which are commercially available from R&D Systems, Inc., 614 McKinley Place NE, Minneapolis, MN 55413.
• Preferably at least 10 ng/ml of each of these growth factors is added, more preferably 10-500 ng/ml.
• the growth factors supplied will determine whether the hemangioblasts become hematopoietic cells or endothelial cells.
• the hemangioblasts can be supplied with a cocktail containing SCF, TPo and FLT3 ; to obtain endothelial cells the hemangioblasts can be supplied with a cocktail containing VEGF .
• Suitable concentrations of SCF, TPo and FLT3 are given above.
• Suitable concentrations of VEGF are from 5 to 100 ng/ml.
• the concentration can be increased, for example, by separating out non-target cells and then placing the remaining target cells in closer proximity to each other, e.g., by decreasing the cross-sectional diameter of the culture vessel. If separation is performed, as discussed above, the duration of the time periods between separations will affect cell proliferation.
• the removal of non-target cells from the culture promotes cell proliferation by reducing build-up of by-products and inhibitors, and thus frequent selection will result in enhanced proliferation of the target population. In most cases, it is preferred that the selection remove substantially all of the non- target cells, as this will minimize inhibition. However, in some cases it may be desirable to allow other populations to proliferate with the target population.
• selection can be eliminated completely if a mixed population can be used or would be desirable in the intended application, e.g., bone marrow restoration where a Graft vs. Leukemia (GVL) type effect is desired.
• Other factors that will influence the composition of the cell population resulting from proliferation include the nutrient media used, the gas tension in the incubator, the seeding density (initial concentration of non-fetal hemangioblasts), and stirring of the culture. These factors could be readily adjusted by one skilled in the art to obtain a desired result.
• a patient requiring immunotherapy has a small volume of blood drawn.
• This blood is then used as described above to produce a pool of autologous or allogeneic non-fetal hemangioblasts, which is administered, using standard methods, to the patient as an immune system booster prior to a treatment damaging the patient's immune system and/or blood forming system (e.g., chemotherapy), and/or as a stimulant to the patient's compromised immune or blood forming system after the treatment.
• Cells are administered to reconstitute portions of the immune system, e.g., bone marrow, using methods described in the literature, e.g., in U.S. Patent Nos.
• a cell sample can be used to produce a pool of a selected population of cells, by first forming a population of non- fetal hemangioblasts and then placing the cells in a culture under conditions selected to promote the proliferation of a desired population of cells using the non-fetal hemangioblasts as precursors .
• Newly formed endothelial cells differentiated from the hemangioblasts can be used in various cell therapies .
• the cells can be used in wound healing, e.g., donor endothelial cells delivered by injection have been found to localize exclusively to neovascular zones of recipient mice. T. Asahara et al . , Isolation of Putative Progenitor Endothelial Cells for Angiogenesis, Science, Vol. 275, p. 964, Feb. 14, 1997.
• Endothelial cells can also be used in gene therapy, for example endothelial cells can be delivered to inhibit vascularization of tumors, e.g., using switchable genes encoding angiogenesis-inhibiting proteins as disclosed in U.S.
• An in vi tro production system would be particularly useful for angiogenic activity assays used in cancer detection, screening of anti-angiogenesis agents for cancer therapeutics, and for graft tissue engineering applications, e.g., grafts for neo- vascularization in the treatment of eschemic cardiovascular diseases, including coronary artery, peripheral artery and cerebral vascular.
• Iscove's Modified Dulbecco's Medium (1MDM) (100 ml) , Pen/Strep (50 ⁇ l) , BSA (50 mg/ml) , Insulin (50 ⁇ g/ml) , Transferrin (1 mg/ml), Low Density Lipoprotein (100 ⁇ l) , 2-Mercapto-Ethanol (7 ⁇ l of 1/100 solution), FLT3 (100 ng/ml), SCF (100 ng/ml), Tpo (100 ng/ml) .
• MDM Iscove's Modified Dulbecco's Medium
• Pen/Strep 50 ⁇ l
• BSA 50 mg/ml
• Insulin 50 ⁇ g/ml
• Transferrin (1 mg/ml)
• Low Density Lipoprotein 100 ⁇ l
• 2-Mercapto-Ethanol 7 ⁇ l of 1/100 solution
• FLT3 100 ng/ml
• SCF 100 ng/
• a Mononuclear Cell Composition (Buffy Coat) using Ficoll-Paque (Pharmacia Biotech) Gradient Centrifugation was prepared and rinsed twice with PBS (without Ca++ or Mg++) .
• the pump was started in the downward direction to allow the sample to run into the matrix of the column. 10.
• PBS plus FBS was added to the top of the column intermittently so as not to allow the sample front to enter the column matrix until 25 mis were collected downstream of the magnet .
• the 25 ml sample containing the target population was centrifuged at 800 RPM for 15 minutes.
• the resulting pellet was resuspended in 1 ml of HBS and transferred to a 1.5 ml eppendorf tube.
• the sample was rinsed twice with repeated centrifugation at 800 RPM and resuspended in 1 ml HBS. 14. Following analysis by flow cytometry the target cells were plated in the growth medium at a concentration of 1000 cells/ml.
• FIG. 5 is a graph showing the growth kinetics of CD34 + /CD38 “ /Lin " and CD34 " /CD38 “ /Lin " target cells over a 30 day culture period. As shown in Fig. 5, the population of CD34 " , Lin " cells continued to expanded throughout the 30 day period.
• Fig. 6 is a group of dot plots showing the population profile of reselected cells prior to subsequent reselection using CD34 vs. Lineage (Lin) markers over a three week period. We observed (a) continual output of lineage positive progeny prior to each selection step; (b) continual amplification of CD34 + , Lin “ cells, and (c) continual amplification of CD34 " , Lin " cells .
• Fig. 7 is a dot plot showing the population profile of initial negatively selected target populations used to inoculated cultures.
• VEGF R CD34 vs. anti-VEGF receptor
• Fig. 8 is a group of dot plots showing the population profile of reselected cells at week three of culture using CD45 vs. MHC I and MHC II and CD34 vs. Flk- 1. Virtually all cells in this culture are CD45+ and a vast gradient exists in MHC expression.
• Fig. 9 is a group of dot plots showing the population profile of reselected cells at week three of selective culture using CXCR4 vs. Flk-1 and Pgp (MDR) vs. Flk-1.
• the circled cells were CD34+ (backgated analysis) and the cells indicated by rectangles were CD34-/CXCR4+/Flk+ and CD34-/Pgp+/Flk+ populations.
• CXCR4 is a chemokine receptor responsible for trafficking engrafting cells into the bone marrow.
• Pgp is the receptor for the Multi-Drug Resistance (MDR) gene product- that corresponds to Hoechst Lo, which has been correlated with CD34- cells in the mouse and rhesus that have high engraftment potential.
• MDR Multi-Drug Resistance

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