US20060013805A1 — Transgenic circulating endothelial cells

Claim 1

Independent Claim 73 words

1 . A process for expanding the population of endothelial cells obtained from peripheral blood comprising culturing, in contact with a collagen I-coated surface, buffy coat cells obtained from peripheral mammalian blood in the presence of a cell culture medium containing an effective amount of vascular endothelial growth factor (VEGF), and which medium is free of bovine brain extract, so as to expand the population of endothelial cells in said buffy coat cells.

Claim 2

Dependent Claim 73 words

1 . A process for expanding the population of endothelial cells obtained from peripheral blood comprising culturing, in contact with a collagen I-coated surface, buffy coat cells obtained from peripheral mammalian blood in the presence of a cell culture medium containing an effective amount of vascular endothelial growth factor (VEGF), and which medium is free of bovine brain extract, so as to expand the population of endothelial cells in said buffy coat cells.

Claim 3

Dependent Claim 13 words

2 . The process of claim 1 wherein the blood is human blood.

Claim 4

Dependent Claim 19 words

3 . The process of claim 1 wherein said cell culture medium comprises heparin, dextran sulfate or mixtures thereof.

Claim 5

Dependent Claim 35 words

4 . The process of claim 1 wherein the buffy coat cells are obtained by washing cells from a buffy coat layer obtained from human blood in cell culture medium comprising 20% human male serum.

Claim 6

Dependent Claim 18 words

5 . The process of claim 1 wherein the cell culture medium comprises human basic fibroblast growth factor.

Claim 7

Dependent Claim 18 words

6 . The process of claim 1 or 5 wherein the cell culture medium comprises insulin-like growth factor.

Claim 8

Dependent Claim 19 words

7 . The process of claim 1 or 5 wherein the cell culture medium contains human epidermal growth factor.

Claim 9

Dependent Claim 28 words

8 . The process of claim 1 wherein the cell culture medium comprises about 0.5-10 vol-% fetal bovine serum and about 95-99.5 vol-% of a cell culture medium.

Claim 10

Dependent Claim 38 words

9 . The process of claim 1 wherein the cultured cells are trypsinized at about 10 3 -fold expansion, collected by centrifugation, resuspended in cell culture medium, and subjected to continued culture in contact with a fibronectin/gelatin-coated surface.

Claim 11

Dependent Claim 15 words

10 . The process of claim 1 wherein the cultured cells are subjected to cryopreservation.

Claim 12

Dependent Claim 26 words

11 . The process of claim 10 wherein the cells are frozen in a cryopreservation medium comprising fetal calf serum containing an effective amount of dimethylsulfoxide.

Claim 13

Dependent Claim 24 words

12 . The process of claim 10 or 11 wherein the cryopreserved cells are thawed and culturing is resumed in said cell culture medium.

Claim 14

Dependent Claim 15 words

13 . The process of claim 1 wherein the expanded population comprises microvascular endothelial cells.

Claim 15

Dependent Claim 23 words

14 . The process of claim 13 wherein the microvascular endothelial cells are CD34 + , CD36 + and express the P1H1 antigen.

Claim 16

Dependent Claim 18 words

15 . A transgenic mammalian endothelial cell comprising an isolated DNA sequence encoding a recombinant Factor VIII protein.

Claim 17

Dependent Claim 18 words

15 . A transgenic mammalian endothelial cell comprising an isolated DNA sequence encoding a recombinant Factor VIII protein.

Claim 18

Dependent Claim 19 words

16 . The transgenic endothelial cell of claim 15 wherein the Factor VIII protein is a hybrid human/porcine protein.

Claim 19

Dependent Claim 47 words

17 . A transgenic endothelial cell which is prepared by a process comprising stably transforming a population of circulating human endothelial cells outgrown from blood with a vector comprising an isolated DNA sequence encoding a preselected protein operably linked to a promoter functional in human endothelial cells.

Claim 20

Dependent Claim 47 words

17 . A transgenic endothelial cell which is prepared by a process comprising stably transforming a population of circulating human endothelial cells outgrown from blood with a vector comprising an isolated DNA sequence encoding a preselected protein operably linked to a promoter functional in human endothelial cells.

Claim 21

Dependent Claim 18 words

18 . The transgenic endothelial cell of claim 17 wherein the DNA sequence encodes a Factor VIII protein.

Claim 22

Dependent Claim 21 words

19 . The transgenic endothelial cell of claim 17 wherein the DNA sequence comprises SEQ ID NO:1 or SEQ ID NO:2.

Claim 23

Dependent Claim 19 words

20 . The transgenic endothelial cell of claim 18 wherein the Factor VIII protein is a hybrid human/porcine protein.

Claim 24

Dependent Claim 19 words

21 . The transgenic endothelial cell of claim 18 wherein the Factor VIII protein is a chimeric human protein.

Claim 25

Dependent Claim 23 words

22 . The transgenic endothelial cell of claim 17 wherein the DNA sequence further comprises a selectable marker gene or a reporter gene.

Claim 26

Dependent Claim 14 words

23 . The transgenic endothelial cell of claim 17 which is prepared by lipofection.

Claim 27

Dependent Claim 25 words

24 . A pharmaceutical composition comprising a population of the transgenic endothelial cells of claim 15 or 17 in combination with a pharmaceutically acceptable carrier.

Claim 28

Dependent Claim 47 words

25 . A method of treating hemophilia comprising introducing an amount of the endothelial cells of claim 15 or 17 into the bloodstream of a mammal afflicted with hemophilia so that an effective amount of a Factor VIII protein is secreted into the bloodstream of said mammal.

Claim 29

Dependent Claim 13 words

26 . The method of claim 25 wherein the mammal is a human.

Claim 30

Dependent Claim 39 words

27 . An implantable medical prosthetic device comprising a surface coated with the endothelial cells of claim 17 wherein the protein is expressed in an amount effective to increase the biocompatibility of said device upon implantation into a mammal.

Claim 31

Dependent Claim 19 words

28 . The device of claim 27 which comprises a plastic surface, a metal surface or a laminate surface.

Claim 32

Dependent Claim 12 words

29 . The device of claim 28 which is a vascular graft.

Claim 33

Dependent Claim 14 words

30 . The device of claim 28 which is a shunt or a stent.

Claim 34

Dependent Claim 12 words

31 . The device of claim 28 which is a heart valve.

Claim 35

Dependent Claim 14 words

32 . The device of claim 28 which is a controlled drug release depot.

Claim 36

Dependent Claim 123 words

33 . A diagnostic method comprising: detecting or determining whether an expanded population of endothelial cells obtained from peripheral blood of a test mammal has an acquired or genetic indication or disease relative to a control expanded population of endothelial cells obtained from a mammal not at risk of the acquired or genetic indication or disease, wherein the expanded cells are obtained by culturing, in contact with a collagen I-coated surface, mononuclear cells from a buffy coat layer obtained from the peripheral blood of the test mammal in the presence of a cell culture medium containing an effective amount of vascular endothelial growth factor (VEGF), which medium is free of bovine brain extract, so as to yield an expanded population of endothelial cells.

Claim 37

Dependent Claim 123 words

33 . A diagnostic method comprising: detecting or determining whether an expanded population of endothelial cells obtained from peripheral blood of a test mammal has an acquired or genetic indication or disease relative to a control expanded population of endothelial cells obtained from a mammal not at risk of the acquired or genetic indication or disease, wherein the expanded cells are obtained by culturing, in contact with a collagen I-coated surface, mononuclear cells from a buffy coat layer obtained from the peripheral blood of the test mammal in the presence of a cell culture medium containing an effective amount of vascular endothelial growth factor (VEGF), which medium is free of bovine brain extract, so as to yield an expanded population of endothelial cells.

Claim 38

Dependent Claim 16 words

34 . The method of claim 33 wherein the indication or disease is a clotting disorder.

Claim 39

Dependent Claim 23 words

35 . The method of claim 33 wherein the indication or disease is associated with a reduction in the activity of an enzyme.

Claim 40

Dependent Claim 14 words

36 . The method of claim 33 wherein the indication or disease is acquired.

Claim 41

Dependent Claim 23 words

37 . The method of claim 33 wherein the indication or disease is associated with expression of a mutant gene in endothelial cells.

Claim 42

Dependent Claim 31 words

38 . The method of claim 33 wherein the indication or disease is associated with altered expression of a gene in endothelial cells of the test mammal relative to the control.

Claim 43

Dependent Claim 13 words

39 . The method of claim 33 wherein the mammal is a human.

Claim 44

Dependent Claim 15 words

40 . The method of claim 33 wherein the cultured cells were subjected to cryopreservation.

Claim 45

Dependent Claim 15 words

41 . The method of claim 33 wherein the expanded population comprises microvascular endothelial cells.

Claim 46

Dependent Claim 23 words

42 . The method of claim 41 wherein the microvascular endothelial cells are CD34 + , CD36 + and express the P1H1 antigen.

Claim 47

Dependent Claim 19 words

43 . The method of claim 33 wherein antibodies were not employed to obtain the buffy coat mononuclear cells.

Transgenic circulating endothelial cells

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