Method for identifying, expanding, and removing adult stem cells and cancer stem cells

Patent metadata

Field	Value
Publication number	US-9833496-B2
Application number	US-201414494511-A
Country	US
Kind code	B2
Filing date	Sep 23, 2014
Priority date	Aug 10, 2007
Publication date	Dec 5, 2017
Grant date	Dec 5, 2017

Abstract

The invention relates to the fields of biochemistry, pharmacy and oncology. The invention particularly relates to the use of novel stem cell markers for the isolation of stem cells. The invention further relates to the obtained stem cells and their use in for example research or treatment, for example, for the preparation of a medicament for the treatment of damaged or diseased tissue.
In one of the embodiments, the invention provides a method for obtaining (or isolating) stem cells comprising optionally preparing a cell suspension from a tissue or organ sample, contacting said cell suspension with an Lgr 6 or 5 binding compound, identify the cells bound to said binding compound, and optionally isolating the stem cells from said binding compound.
The invention further relates to means suitable for cancer treatment and even more specific for the treatment of cancer by eradicating cancer stem cells.

First claim

The invention claimed is:

1. A genetically modified Lgr5 positive stem cell produced by a method comprising:
providing a cell suspension;
contacting a cell suspension with an antibody specific for Lgr5 so as to bind the antibody, antibody derivative or antibody fragment specific for Lgr5 to Lgr5 positive cells in the cell suspension;
at least partially purifying cells bound to the antibody, antibody derivative or antibody fragment specific for Lgr5 from the cell suspension so as to obtain an Lgr5+stem cell, and
genetically modifying the obtained Lgr5+stem cell by introducing into the cell:
(a) a non-native nucleotide sequence, and/or
(b) a coding sequence at a non-specific, non-native genomic location, and/or
(c) a non-native promoter sequence that alters the expression of one or more genes, and/or
(d) a nucleic acid encoding a non-native reporter protein, and/or
(e) a multi-cistron coding nucleic acid comprising a sequence encoding at least one non-native protein,
wherein the genetically modified Lgr5 positive stem cell is in vitro.

2. A stem cell, comprising Lgr5 in its cell membrane, that is genetically modified by:
(a) introduction of a non-native nucleotide sequence into the cell, and/or
(b) insertion of a coding sequence at a non-specific, non-native genomic location, and/or
(c) introduction of a non-native promoter sequence that alters the expression of one or more genes, and/or
(d) introduction of a nucleic acid encoding a non-native reporter protein, and/or
(e) introduction of multi-cistron coding nucleic acid comprising a sequence encoding at least one non-native protein, wherein the stem cell is in vitro.

3. The stem cell according to claim 2 , wherein the stem cell is a multipotent adult stem cell.

4. The stem cell according to claim 2 , wherein the stem cell is an epithelial stem cell.

5. The stem cell according to claim 2 , wherein the stem cell is an intestine, brain, liver, retina, stomach, pancreas, ovary, adrenal medulla, skin, bladder, bone, connective tissue, ear, muscle, prostate, placenta, uterus, or breast stem cell.

6. The stem cell according to claim 5 , which is a stem cell of the small intestine or colon or which is a rectal stem cell.

7. The stem cell according to claim 2 , wherein the stem cell is bound to an antibody, antibody derivative or antibody fragment specific for Lgr5.

8. The stem cell according to claim 2 , wherein the stem cell is a cancer stem cell.

9. An isolated collection of stem cells, comprising stem cells according to claim 2 .

10. The isolated collection of stem cells according to claim 9 , which express Lgr5 at a higher level than in transit amplifying cells as determined by in situ hybridization.

11. The isolated collection of stem cells according to claim 9 , further comprising their immediate transit-amplifying daughter cells.

12. The isolated collection of stem cells according to claim 9 , wherein at least 50% of said stem cells are cancer stem cells that are able to recapitulate the generation of a continuously growing tumour.

13. A culture of stem cells according to claim 2 .

14. The culture of stem cells according to claim 13 , wherein the culture is in serum-free media.

15. The culture of stem cells according to claim 13 , further comprising an antibody specific for Lgr5 or an antigen-binding fragment of said Lgr5 antibody.

16. The culture of stem cells according to claim 13 , further comprising Matrigel® (basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells).

17. A composition comprising an stem cell according to claim 2 and a candidate therapeutic compound.

18. A medicament for treating damaged or diseased tissue, comprising stem cells according to claim 2 .

19. A composition comprising the stem cell according to claim 1 and Matrigel® (basement membrane preparation extracted from Engelbreth-Holm-S warm (EHS) mouse sarcoma cells).

20. The stem cell according to claim 1 , wherein the non-native nucleotide sequence encodes a non-native protein and/or the multi-cistron coding nucleic acid comprises an internal ribosomal entry site (IRES).

21. The stem cell according to claim 2 , wherein the non-native nucleotide sequence encodes a non-native protein and/or the multi-cistron coding nucleic acid comprises an internal ribosomal entry site (IRES).

22. The isolated collection of stem cells according to claim 10 , wherein the transit amplifying cells are immediate transit amplifying daughter cells.

23. An isolated population of cells comprising a genetically modified Lgr5 positive stem cell, wherein the genetically modified Lgr5 positive stem cell is produced by a method comprising:
providing a cell suspension;
contacting a cell suspension with an antibody specific for Lgr5 so as to bind the antibody, antibody derivative or antibody fragment specific for Lgr5 to Lgr5 positive cells in the cell suspension;
at least partially purifying cells bound to the antibody, antibody derivative or antibody fragment specific for Lgr5 from the cell suspension so as to obtain an Lgr5+stem cell, and
genetically modifying the obtained Lgr5+stem cell by introducing into the cell:
(a) a non-native nucleotide sequence, and/or
(b) a coding sequence at a non-specific, non-native genomic location, and/or
(c) a non-native promoter sequence that alters the expression of one or more genes, and/or
(d) a nucleic acid encoding a non-native reporter protein, and/or
(e) a multi-cistron coding nucleic acid comprising a sequence encoding at least one non-native protein.

24. An isolated population of cells comprising a stem cell, wherein the stem cell comprises Lgr5 in its cell membrane and wherein the stem cell is genetically modified by:
(a) introduction of a non-native nucleotide sequence into the cell, and/or
(b) insertion of a coding sequence at a non-specific, non-native genomic location, and/or
(c) introduction of a non-native promoter sequence that alters the expression of one or more genes, and/or
(d) introduction of a nucleic acid encoding a non-native reporter protein, and/or
(e) introduction of multi-cistron coding nucleic acid comprising a sequence encoding at least one non-native protein.

Assignees

• Koninklijke Nederlandse Akademie Van Wetenschappen

Inventors

• Clevers Johannes C
• Barker Nicholas
• Haegebarth Andrea
• Van De Wetering Marcus Lambertus

Classifications

• A61K38/177 Primary

  Receptors; Cell surface antigens; Cell surface determinants · CPC title

• A61K45/06

  Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca · CPC title

• C12N5/068 Primary

  Stem cells; Progenitors · CPC title

• A61P35/00

  Antineoplastic agents · CPC title

• G01N33/5011

  for testing antineoplastic activity · CPC title