GENERATION OF ADIPOSE TISSUE AND ADIPOCYTES

• US 20100015104A1
• Filed: 07/25/2007
• Published: 01/21/2010
• Est. Priority Date: 07/26/2006
• Status: Abandoned Application

• Current Assignee: Lorem Vascular Pte. Ltd.
• Original Assignee: Cytori Therapeutics Incorporated
• Inventors: DeEmidio, Michael, Fraser, John K.
• Application Number:
  US12/375,005
• Publication Number:
  US 20100015104A1
• Time in Patent Office: Days
• US Class Current: 424/93.700
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: C12N 2501/599 with CD designations not provided for elsewhere
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 5/0653 Adipocytes; Adipose tissue

Abstract

The invention provides novel methods by which adipose tissue, preadipocytes, and adipocytes can be generated for research purposes, and methods for identifying cell populations that can proliferate and differentiate into adipocytes in vivo. The invention further provides a means for the in vivo derivation of “designer” or “customized” adipose tissue, preadipocytes, and adipocytes. Also provided are methods for identifying agents that affect adipocytes and adipose tissue, as well as the agents themselves. In particular, the present invention allows for creation of tissues and cells that can be used to screen for agents useful for treating human disorders associated with adipose tissue, including obesity, metabolic syndrome, and diabetes.

Claims

1. A method for identifying an isolated population of adipose-derived regenerative cells capable of generating adipocytes or adipose tissue in a subject, comprising:

a. obtaining isolated adipose-derived regenerative cells from a subject;

b. sorting said isolated adipose-derived regenerative cells into at least two different cell populations according to cell surface markers present on said cells;

c. providing at least one of said at least two different cell populations to at least one host animal; and

d. determining the presence, absence, quality, or amount of adipocytes or adipose tissue generated by the at least one of said two different cell populations provided in step (c) in said at least one host animal.

2. A method for identifying a molecule that modulates a biological property of adipocytes or adipose tissue in a subject, comprising:

a. obtaining isolated adipose-derived regenerative cells from a subject;

b. providing said isolated adipose-derived regenerative cells to at least one host animal;

c. determining the presence, absence, quality, or amount of adipocytes or adipose tissue generated by said isolated adipose-derived regenerative cells in said at least one host animal;

d. providing a candidate molecule that modulates a biological property of adipocytes or adipose tissue to said host animal; and

e. determining whether said candidate molecule modulates a biological property of adipocytes or adipose tissue in said host animal.

3. A method for identifying a molecule that modulates the activity of a toxicant on adipocytes or adipose tissue in a subject, comprising:

a. obtaining isolated adipose-derived regenerative cells from a subject;

b. providing said isolated adipose-derived regenerative cells to at least one host animal;

c. determining the presence, absence, quality, or amount of adipocytes or adipose tissue generated by said isolated adipose-derived regenerative cells in said at least one host animal;

d. providing said toxicant to said at least one host animal;

e. providing a candidate molecule that modulates the activity of a toxicant on adipocytes or adipose tissue to said host animal; and

f. determining whether said candidate molecule modulates the activity of a toxicant on adipocytes or adipose tissue in said host animal.

4. A method of making an adipose-derived regenerative cell medicament comprising:

a. obtaining isolated adipose-derived regenerative cells from a subject;

b. sorting said isolated adipose-derived regenerative cells into at least two different cell populations according to cell surface markers present on said cells;

c. providing at least one of said at least two different cell populations to at least one host animal;

d. determining the presence, absence, quality, or amount of adipocytes or adipose tissue generated by the at least one of said two different cell populations provided in step (c) in said at least one host animal; and

e. incorporating a cell population that is determined to generate adipocytes or adipose tissue in step (d) into a medicament.

5. A method of adipose-derived regenerative cell transplantation comprising:

a. obtaining isolated adipose-derived regenerative cells from a subject;

b. sorting said isolated adipose-derived regenerative cells into at least two different cell populations according to cell surface markers present on said cells;

c. providing at least one of said at least two different cell populations to at least one host animal;

d. determining the presence, absence, quality, or amount of adipocytes or adipose tissue generated by the at least one of said two different cell populations provided in step (c) in said at least one host animal;

e. incorporating a cell population that is determined to generate adipocytes or adipose tissue in step (d) into a medicament; and

f. providing said medicament to a patient that is identified as one in need of adipose-derived regenerative cell transplantation.

6. The method of claim 1, wherein the host animal or subject or both are immunotolerant, syngenic, or lipoatropic.

7. The method of claim 1, wherein the presence, absence, quality, or amount of adipocytes or adipose tissue generated by at least two different cell populations sorted according to cell surface markers are compared in either the same or different host animals.

8. The method of claim 1, wherein in a first model, the presence, absence, quality, or amount of adipocytes or adipose tissue generated by at least one of the at least two different cell populations sorted according to cell surface markers is compared to a second model, wherein the presence or absence of adipocytes or adipose tissue generated by the isolated adipose-regenerative cells prior to cell sorting in either the same or different host animals are determined.

9. The method of claim 2, wherein said isolated adipose-derived regenerative cells are sorted into at least two different cell populations according to cell surface markers present on said cells and at least one of said at least two different sorted cell populations are provided to at least one host animal to which the candidate molecule or the candidate molecule and toxicant are provided.

10. The method of claim 3, wherein said isolated adipose-derived regenerative cells are sorted into at least two different cell populations according to cell surface markers present on said cells and at least two different sorted cell populations are provided to at least one host animal to which the candidate molecule or the candidate molecule and toxicant are provided, and, optionally, the modulation of the biological property of adipocytes or adipose tissue or the modulation of the activity of the toxicant at the sites of introduction of said at least two different sorted cell populations are compared.

11. The method of claim 2, wherein in a first model, said isolated adipose-derived regenerative cells are sorted into at least two different cell populations according to cell surface markers present on said cells, at least one of the at least two different sorted cell populations are provided to at least one host animal to which the candidate molecule or the candidate molecule and toxicant are provided and in a second model, a portion of said isolated adipose-derived regenerative cells are provided to either the same or a different host animal to which the candidate molecule or the candidate molecule and toxicant are provided, and, optionally, the modulation of the biological property of adipocytes or adipose tissue or the modulation of the activity of the toxicant in the two models are compared.

12. The method of claim 1, wherein the isolated adipose-derived regenerative cells are from a human.

13. The method of claim 1, wherein the host animal is a human.

14. The method of claim 1, wherein the host animal is a mouse.

15. The method of claim 1, wherein the subject from which the isolated adipose-derived regenerative cells are obtained and host animal, which receives said isolated adipose-derived regenerative cells are the same species.

16. The method of claim 1, wherein the subject from which the isolated adipose-derived regenerative cells are obtained and host animal, which receives said isolated adipose-derived regenerative cells are the same individual.

17. The method of claim 1, wherein the isolated adipose-derived regenerative cells and/or a sorted cell population are genetically modified prior to providing said the isolated adipose-derived regenerative cells and/or a sorted cell population to said host animal.

18. The method of claim 17, wherein said isolated adipose-derived regenerative cells and/or a sorted cell population is genetically modified with a marker gene such as, GFP, luciferase, or B-gal.

19. The method of claim 1, wherein said isolated adipose-derived regenerative cells and/or a sorted cell population are isolated while maintaining a closed/sterile fluid pathway.

20. The method of claim 1, wherein said sorting utilizes flow cytometry.

21. The method of claim 1, wherein said sorting is based on analysis of at least two cell surface markers, at least three cell surface markers, at least four cell surface markers, at least five cell surface markers, or at least six cell surface markers.

22. The method of claim 1, wherein said at least two different cell populations are provided to the same host animal at different locations.

23. The method of claim 1, wherein said at least two different cell populations are provided to different host animals of the same species.

24. The method of claim 1, wherein said at least two different cell populations are provided to different host animals of different species.

25. The method of claim 1, wherein the presence or absence of adipocytes or adipose tissue in said at least one host animal is determined by measuring the appearance, size, morphology, or a biochemical marker of said adipocytes or adipose tissue.

26. The method of claim 1, further comprising determining the presence, absence, quality, or amount of angiogenesis, arteriogenesis, or lymphangiogenesis in said host animal.

27. The method of claim 26, wherein said determination of the presence, absence, quality, or amount of adipocytes or adipose tissue in said at least one host animal is determined by histology.

28. The method of claim 26, wherein said determination of the presence, absence, quality, or amount of adipocytes or adipose tissue in said at least one host animal is determined by staining.

29. The method of claim 26, wherein said determination of the presence, absence, quality, or amount of adipocytes or adipose tissue in said at least one host animal is determined by detection of a biological marker without sacrificing the host animal.

30. The method of claim 26, wherein said determination of the presence, absence, quality, or amount of adipocytes or adipose tissue in said at least one host animal is determined by detection of a GFP without sacrificing the host animal.

31. The method of claim 1, wherein at least one of the two different cell populations that are provided to at least one host animal expresses CD73, does not express CD45 or CD31, and expresses low levels of or no CD90.

32. The method of claim 2, wherein said modulation of activity is an up-regulation of activity.

33. The method of claim 3, wherein said modulation of activity is a down-regulation of activity.

34. The method of claim 2, wherein said candidate molecule is a hormone, an adipokine, an angiogenic modulating molecule, a lymphangiogenic modulating molecule, an immunomodulatory molecule, or an arteriogenic modulatory molecule.

35. The method of claim 5, wherein said patient that is identified as one in need of adipose-derived regenerative cell transplantation is a patient in need or that desires soft tissue implantation or regeneration.

36. The method of claim 35, wherein the cell population provided to said patient expresses CD73, does not express CD45 or CD31, and that expresses low levels of CD90 or no CD90.

37. The method of claim 5, wherein said patient that is identified as one in need of adipose-derived regenerative cell transplantation is a patient with obesity, obesity metabolic syndrome, or diabetes.

38. The method of claim 37, wherein the cell population provided to said patient expresses CD73, does not express CD45 or CD31, and that expresses low levels of CD90 or no CD90.

39. The method of claim 5, wherein said patient that is identified as one in need of adipose-derived regenerative cell transplantation is a patient with a cardiovascular disorder or peripheral vascular disease.

40. The method of claim 39, wherein the cell population provided to said patient expresses CD73, does not express CD45 or CD31, and that expresses low levels of CD90 or no CD90.

41. (canceled)