Regulating stem cells

• US 9,234,173 B2
• Filed: 08/20/2013
• Issued: 01/12/2016
• Est. Priority Date: 03/08/2006
• Status: Active Grant

• Current Assignee: Kwalata Trading Ltd.
• Original Assignee: Kwalata Trading Ltd.
• Inventors: Porat, Yael, Fulga, Valentin, Porozov, Svetlana, Belleli, Adina
• Primary Examiner(s): Ware, Debbie K
• Assistant Examiner(s): Srivastava, Kailash C
• Application Number:
  US13/970,807
• Publication Number:
  US 20130345083A1
• Time in Patent Office: 875 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 35/44 Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
• CPC Class Codes: C12N 2501/14 Erythropoietin [EPO]
• CPC Class Codes: C12N 2501/165 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: C12N 2501/23 Interleukins [IL]
• CPC Class Codes: C12N 2501/33 Insulin together with transferrin C12N2500/25; Insulin-like growth factors C12N2501/105
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/392 Sexual steroids
• CPC Class Codes: C12N 2501/599 with CD designations not provided for elsewhere
• CPC Class Codes: C12N 2501/91 Heparin
• CPC Class Codes: C12N 2506/11 from blood or immune system cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 2533/54 Collagen; Gelatin
• CPC Class Codes: C12N 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0634 Cells from the blood or the immune system
• CPC Class Codes: C12N 5/0692 Stem cells; Progenitor cells; Precursor cells

Abstract

Provided are methods for producing progenitor/precursor cells from a population of initiating cells (ICP) that have a density of less than 1.072 g/ml and at least 25% of which are CD31Bright by in vitro stimulating the ICP with different factors.

Claims

1. A method of making a composition comprising:

obtaining an initiating cell population (ICP) of at least ten thousand cells that have a density of less than 1.072 g/ml and at least 25% of which are CD31Bright; and

in vitro stimulating the ICP by culturing the ICP in the presence of one or more factors selected from the group consisting of;

autologous serum, vascular endothelial growth factor (VEGF), heparin, insulin-like growth factor (IGF), basic fibroblast growth factor (b-FGF), epidermal growth factor (EGF), and ascorbic acid, to differentiate into a progenitor/precursor cell population (PCP) comprising a subpopulation of cells that stain as CD31Bright, demonstrate uptake of Ac-LDL+ and secrete interleukin-8 (IL-8) and angiogenin, wherein the sub-population of cells comprises at least 10% of cells in the PCP, to provide for said composition.

2. The method according to claim 1, wherein at least 30% of the cells of the ICP are CD31Bright, and wherein stimulating the ICP comprises stimulating the ICP of which at least 30% of the cells are CD31Bright.

3. The method according to claim 1, wherein the ICP includes at least 5 million cells that have a density of less than 1.062 g/ml, at least 1% of which are CD34+CD45−

/Dim, and wherein stimulating the ICP comprises stimulating the ICP that has the at least 5 million cells that have a density of less than 1.062 g/ml.

4. The method according to claim 1, comprising applying cells extracted from a mammalian donor to one or more gradients suitable for selecting cells having a density of less than 1.072 g/ml, and deriving the ICP from the cells applied to the gradient.

5. The method according to claim 1, comprising identifying the PCP as being suitable for therapeutic implantation in response to an assessment that the PCP includes at least 1 million PCP cells.

6. The method according to claim 1, comprising identifying the PCP as being suitable for therapeutic implantation in response to an assessment that the PCP includes at least 1 million angiogenic cell precursors (ACPs).

7. The method according to claim 1, wherein stimulating the ICP comprises culturing the ICP in a culture medium comprising at least 10% serum, for a duration of between 1 and 120 days.

8. The method according to claim 1, comprising characterizing the PCP as including angiogenic cell precursors (ACPs), in response to an evaluation of at least one feature selected from the group consisting of:

a phenotypical feature of cells in the PCP, a genotypical feature of cells in the PCP, and a physiological feature of cells in the PCP.

9. The method according to claim 8,

wherein the phenotypical feature includes a morphological feature selected from the group consisting of:

a cell size larger than 20 um, an elongated cell, a spindle-shaped cell, an irregularly-shaped cell, a granulated cell, a cell including an enlarged dark nucleus, a multinuclear cell, a cell including flagella-like structures, a cell including pseudopodia, and a cell having a polygonal shape; and

wherein characterizing the PCP comprises characterizing the PCP in response to an evaluation of the selected morphological feature.

10. The method according to claim 8, wherein characterizing the PCP comprises characterizing the PCP in response to an identification in the PCP of a feature selected from the group consisting of:

CD31, CD31Bright, CD34, CD117, CD133, Tie-2, CD34+CD133+, KDR, CD34+KDR+, CD144, von Willebrand Factor, SH2 (CD105), SH3, fibronectin, collagen type I, collagen type III, collagen type IV, ICAM type 1, ICAM type 2, VCAM1, vimentin, BMP-R IA, BMP-RII, CD44, integrin b1, aSM-actin, MUC18, and CXCR4.

11. The method according to claim 8, wherein characterizing the PCP comprises assessing secretion by the PCP of a molecule selected from the group consisting of:

IL-8, angiogenin, VEGF, MMP2, and MMP9.

12. The method according to claim 8, wherein characterizing the PCP comprises culturing at least a portion of the PCP on a membrane, and identifying a tendency of the at least a portion of the PCP to migrate toward a chemoattractant selected from the group consisting of:

bFGF, VEGF, SCF, G-CSF, GM-CSF, IL-8, and SDF-1.

13. The method according to claim 1, wherein stimulating the ICP comprises incubating the ICP in a container having a surface comprising a growth-enhancing factor.

14. The method according to claim 13, wherein the growth-enhancing factor comprises a factor selected from the group consisting of:

collagen, plasma, fibronectin, a growth factor, tissue-derived extra cellular matrix, and an antibody to a stem cell surface receptor.

15. The method according to claim 1, comprising:

during a hypoxic time period lasting at least 2 hours, culturing the ICP under hypoxic conditions; and

during a non-hypoxic time period lasting at least 1 day, culturing the ICP under non-hypoxic conditions.

16. The method according to claim 15, wherein the hypoxic and non-hypoxic time-periods are within a culturing time period lasting less than 30 days, and wherein culturing the ICP under hypoxic conditions comprises culturing the cells under hypoxic conditions during a first two days of the culturing time period.

17. The method according to claim 1, wherein stimulating comprises co-culturing the ICP with a sample tissue.

18. The method according to claim 17, wherein co-culturing comprises:

utilizing the sample tissue to produce a conditioned medium; and

co-culturing the ICP with the sample tissue in the conditioned medium.