3D trophoblast matrix for preparing organ-specific stem cells

• US 9,102,914 B2
• Filed: 02/03/2011
• Issued: 08/11/2015
• Est. Priority Date: 02/03/2011
• Status: Expired due to Fees

• Current Assignee: Empire Technology Development LLC (Allied Inventors Management, LLC)
• Original Assignee: Empire Technology Development LLC (Allied Inventors Management, LLC)
• Inventors: Gu, Yansong
• Primary Examiner(s): Paras, Jr., Peter
• Assistant Examiner(s): SGAGIAS, MAGDALENE K
• Application Number:
  US13/257,949
• Publication Number:
  US 20120202261A1
• Time in Patent Office: 1,650 Days
• US Class Current: 1/1
• CPC Class Codes: C12N 11/04 entrapped within the carrier, e.g. gel or hollow fibres
• CPC Class Codes: C12N 11/096 Polyesters; Polyamides
• CPC Class Codes: C12N 11/10 the carrier being a carbohydrate
• CPC Class Codes: C12N 2501/00 Active agents used in cell culture processes, e.g. differentation
• CPC Class Codes: C12N 2502/025 extra-embryonic cells, e.g. amniotic epithelium, placental cells, Wharton's jelly
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2533/30 Synthetic polymers
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/06 Animal cells or tissues; Human cells or tissues preservation of living cells or tissues A01N1/02
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0657 Cardiomyocytes; Heart cells

Abstract

This disclosure relates generally to methods, apparatuses, and cellular compositions and cellular products that use a unique trophoblast-containing 3D matrix as a compartmental chamber for growing embryoid bodies that can be induced to differentiate into organ-specific cell types.

Claims

1. A method for creating lineage-specific stem cells, comprising:

a) seeding human embryonic stem cells (hESCs) into a trophoblast-containing three-dimensional matrix;

b) growing an embryoid body from the human embryonic stem cells within the trophoblast-containing matrix;

c) transferring the trophoblast-matrix containing the embryoid body from step (b) into a new culture medium that contains at least one differentiation agent that causes the ES cells to become committed lineage-specific stem cells; and

d) transferring the trophoblast-matrix containing the embryoid body from step (c) into a new culture medium that contains at least one chemoattractant, wherein at least one committed lineage-specific stem cell migrates out of the embryoid body and the trophoblast-containing matrix in response to the chemoattractant.

2. The method of claim 1, further comprising encapsulating human embryonic stem cells in a hydrogel.

3. The method of claim 1, further comprising degenerating the structure of the embryoid body.

4. The method of claim 3, wherein degenerating the structure of the embryoid body comprises digesting the trophoblast-containing matrix comprising the embryoid body.

5. The method of claim 4, further comprising isolating lineage-specific stem cells obtained from the degenerated embryoid body.

6. The method of claim 2, wherein the hydrogel comprises a natural or synthetic polymer.

7. The method of claim 6, wherein the hydrogel comprises a polysaccharide, a peptide, a proteoglycan, or a combination thereof.

8. The method of claim 2, wherein the hydrogel comprises a polymer selected from the group consisting of poly(glycolic acid), poly(lactic acid), poly(glycolic acid-co-lactic acid), collagen, laminin, hyaluronan, alginate, chitosan, silk fibrils, poly(vinyl alcohol), poly(2-hydroxylethyl methacrylate), polyethylene terephthalate, agarose, methylcellulose, fibrin, and a combination thereof.

9. The method of claim 2, wherein the trophoblast-containing matrix is embedded in a hydrogel that is in a well of a 96-well plate.

10. The method of claim 2, wherein the step of transferring the trophoblast-matrix containing the embryoid body to a new culture medium comprises:

(i) dissolving the hydrogel and

(ii) transferring the trophoblast-containing matrix containing the embryoid body into a 96-well plate that is either (a) pre-filled with a hydrogel that comprises at least one differentiation agent for promoting the creation of lineage-specific stem cells, or (b) coated with at least one differentiation agent for promoting the creation of lineage-specific stem cells.

11. The method of claim 10, wherein the differentiation agent is a chemokine or morphogen.

12. The method of claim 1, wherein the trophoblasts are cytotrophoblast cells.

13. The method of claim 1, wherein 10-100,000 human embryonic stem cells are seeded into the trophoblast-containing matrix.

14. The method of claim 1, wherein the differentiation agent promotes the creation of neural stem cells, oligodendrocyte cells, mast cells, hemato-lymphoid cells, epithelial cells, mammary stem cells, mesenchymal stem cells, olfactory stem cells, and testicular stem cells.

15. The method of claim 10, wherein multiple wells of the 96-well plate each contain a trophoblast-containing matrix seeded with embryonic stem cells, and each matrix, when it comprises a grown embryoid body, is simultaneously or subsequently transferred to a well plate that comprises at least one differentiation agent that promotes creation of the same or different lineage-specific stem cell.

16. The method of claim 1, wherein the trophoblast containing-matrix is transferred to a new culture medium once the embryoid body develops beating cardiomyocytes.