Isolated mesenchymal progenitor cells and extracellular matrix produced thereby

Patent metadata

Field	Value
Publication number	US-9144585-B2
Application number	US-201113812552-A
Country	US
Kind code	B2
Filing date	Jul 27, 2011
Priority date	Jul 27, 2010
Publication date	Sep 29, 2015
Grant date	Sep 29, 2015

Abstract

Provided are methods of isolating an extracellular matrix from mesenchymal progenitor cells which are characterized by a reduced differentiation ability into an adipogenic lineage as compared to adipose-derived mesenchymal stem cells. Also provide isolated extracellular matrix and hybrid devices comprising electrospun elements and extracellular matrix which can be used for various tissue regeneration, repair and reconstruction surgeries.

First claim

What is claimed is:

1. A method of isolating an extracellular matrix, comprising:
(a) obtaining induced pluripotent stem cells (iPSCs) derived from plucked human hair follicle keratinocytes (HFKTs);
(b) generating embryoid bodies (EBs) from said iPSCs;
(c) generating single cells from adherent cells of said EBs, and
(d) culturing said single cells in a culture medium which comprises ascorbic acid and does not comprise dexamethasone to thereby obtain a population of mesenchymal progenitor cells which comprises at least 70% CD105+ cells, wherein said mesenchymal progenitor cells exhibit at least 70% reduced differentiation potential into an adipogenic lineage as compared to differentiation of mesenchymal stem cells from an adult adipose source under identical assay conditions as analyzed after 28-30 days of culturing in a medium which comprises 10 −6 M dexamethasone and indomethacin; and subsequently,
(e) culturing said mesenchymal progenitor cells produced in step (d) under conditions which induce production of extracellular matrix from said mesenchymal progenitor cells, and subsequently,
(f) isolating the extracellular matrix produced by said mesenchymal progenitor cells,
thereby isolating the extracellular matrix.

2. The method of claim 1 , further comprising decellularizing said extracellular matrix.

3. The method of claim 1 , wherein said single cells from adherent cells of said EBs are obtained by:
(a) dissociating the EBs to cell aggregates,
(b) culturing said cell aggregates on a low-adhesive surface so as to select a population of adherent cells, and
(c) dissociating said adherent cells to single cells.

4. The method of claim 1 , wherein said EBs are 8-14 day-old human EBs.

5. The method of claim 3 , wherein each of said aggregates comprises about 10-30 cells.

6. The method of claim 3 , wherein said dissociating said EBs into said aggregates is effected using Collagenase B.

7. The method of claim 3 , wherein said adherent cells are expanded by at least 2 fold within 2-3 days of culturing in said culture medium.

8. The method of claim 1 , wherein differentiation into an osteogenic lineage of said mesenchymal progenitor cells is increased by at least 50% as compared to differentiation of mesenchymal stem cells from an adult adipose source under identical assay conditions.

9. The method of claim 1 , wherein said culturing in step (e) is performed on an electrospun element.

10. The method of claim 1 , wherein at least 70% of said population of mesenchymal progenitor cells are CD105+/CD90+.

11. The method of claim 1 , wherein at least 70% of said population of mesenchymal progenitor cells are characterized by a CD105+/CD90+/CD73+/CD44+/CD29+ signature.

12. The method of claim 1 , wherein at least 70% of said population of mesenchymal progenitor cells are characterized by a CD105+/CD45−/CD34− signature.

13. The method of claim 1 , wherein said mesenchymal progenitor cells maintain the ability to form extracellular matrix for at least 8 passages.

14. The method of claim 1 , wherein said culturing in step (d) is performed for 1-15 passages.

15. The method of claim 1 , wherein said iPSCs are lentiviral vector-free iPSCs.

Assignees

• Itskovitz-Eldor Joseph
• Shtrichman Ronit
• Zussman Eyal
• Barak Efrat
• Technion Res & Dev Foundation

Inventors

• Itskovitz-Eldor Joseph
• Shtrichman Ronit
• Zussman Eyal
• Barak Efrat

Classifications

• C12N5/0662

  Stem cells · CPC title

• C12N2506/02

  from embryonic cells · CPC title

• A61K35/12 Primary

  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 title

• C12N2509/00

  Methods for the dissociation of cells, e.g. specific use of enzymes · CPC title

• C12N2506/45

  from artificially induced pluripotent stem cells · CPC title