Methods of isolating and culturing stem cells

• US 8,940,294 B2
• Filed: 03/01/2013
• Issued: 01/27/2015
• Est. Priority Date: 03/02/2012
• Status: Active Grant

• Current Assignee: Biotissue Holdings Incorporated
• Original Assignee: Tissuetech, Inc.
• Inventors: Tseng, Scheffer, Chen, Szu-Yu, Zhang, Suzhen
• Primary Examiner(s): Carlson, Karen Cochrane
• Assistant Examiner(s): MOSS, NATALIE M
• Application Number:
  US13/782,968
• Publication Number:
  US 20130251684A1
• Time in Patent Office: 697 Days
• Field of Search: 424/93.7, 435/325
• US Class Current: 424/93.7
• CPC Class Codes: C12N 2500/34 Sugars
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/235 Leukemia inhibitory factor [LIF]
• CPC Class Codes: C12N 2501/40 Regulators of development
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2533/80 Hyaluronan
• CPC Class Codes: C12N 2533/90 Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
• CPC Class Codes: C12N 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0665 Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
• CPC Class Codes: C12N 5/0667 Adipose-derived stem cells [ADSC]; Adipose stromal stem cells

Abstract

Provided herein are methods of isolating and expanding a plurality of multipotent stem cells. Also described are methods of expanding stem cells on a substrate comprising an HC-HA complex. Also described are isolated and expanded stem cells produced by the methods and uses thereof, including stem cell therapy, as niche cells for supporting other types of stem cells, or as bioreactors for the production of HC-HA complexes. Also described are uses of HC-HA complexes as a carrier for stem cells.

Claims

1. A method for isolating an E-cadherin positive stem cell, comprising:

(a)removing all epithelial cells from an isolated mixed cell population comprising one or more stem cells and one or more non-stem cells; and

(b)after removal of all of the epithelial cells contacting the remaining cells of the mixed cell population with an agent that binds to E-cadherin, thereby isolating an E-cadherin positive stem cell from the mixed cell population.

2. The method of claim 1, wherein the agent that binds to E-cadherin is an antibody.

3. The method of claim 1, further comprising isolating the E-cadherin positive stem cell by fluorescence activated cell sorting or magnetic activated cell sorting.

4. The method of claim 1, wherein the mixed cell population comprises an embryonic stem cell, an adult stem cell, a fetal stem cell, or an induced pluripotent stem cell.

5. The method of claim 1, wherein the mixed cell population comprises a limbal stromal niche cell, an umbilical cord stem cell, an amniotic membrane stem cell or an adipose stem cell.

6. The method of claim 1, further comprising isolating the isolated mixed cell population from an umbilical cord.

7. The method of claim 6, wherein the umbilical cord is a human, non-human primate, cow or pig umbilical cord.

8. The method of claim 6, further comprising:

(a) mechanically or enzymatically removing all epithelial cells from the umbilical cord, thereby producing epithelial free-umbilical cord tissue; and

(b) contacting the epithelial free-umbilical cord tissue with collagenase for a period of time sufficient to release one or more stem cells from other bound cells and components of the stromal matrix of the epithelial free-umbilical cord tissue, thereby producing the isolated mixed cell population.

9. The method of claim 8, further comprising removing an umbilical cord blood vessel from the epithelial free-umbilical cord tissue prior to contacting the epithelial free-umbilical cord tissue with collagenase.

10. The method of claim 1, further comprising isolating the isolated mixed cell population from adipose tissue.

11. The method of claim 10, further comprising digesting the adipose tissue with collagenase, thereby producing collagenase-digested adipose tissue, thereby producing the isolated mixed cell population.

12. The method of claim 11, further comprising digesting the adipose tissue with collagenase in modified ESC medium.

13. The method of claim 11, further comprising fractionating the collagenase-digested adipose tissue by centrifugation, thereby producing a floating cell fraction (FC) and a sedimented stromal vascular fraction (SVF).

14. The method of claim 13, further comprising selecting the FC as the isolated mixed cell population.

15. The method of claim 13, further comprising selecting the sedimented SVF as the isolated mixed cell population.

16. The method of claim 13, further comprising filtering the sedimented SVF on a mesh filter, thereby producing a filtered SVF and a remaining cell fraction (RC).

17. The method of claim 16, further comprising selecting the filtered SVF as the isolated mixed cell population.

18. The method of claim 16, further comprising selecting the RC as the isolated mixed cell population.

19. The method of claim 16, wherein the filter has a pore size of about 40 μ

m to about 250 μ

m.

20. The method of claim 1, further comprising isolating the isolated mixed cell population from amniotic membrane.

21. The method of claim 20, further comprising:

(a) contacting the amniotic membrane with collagenase, thereby producing collagenase-digested amniotic membrane; and

(b) contacting the collagenase-digested amniotic membrane with dispase, thereby producing the isolated mixed cell population.

22. The method of claim 20, further comprising:

(a) contacting the amniotic membrane with dispase, thereby producing dispase-digested amniotic membrane; and

(b) contacting the dispase-digested amniotic membrane with collagenase, thereby producing the isolated mixed cell population.

23. A method for isolating an E-cadherin positive stem cell, comprising contacting an isolated mixed cell population comprising one or more stem cells and one or more non-stem cells with an agent that binds to E-cadherin, thereby isolating an E-cadherin positive stem cell from the mixed cell population, wherein the isolated mixed cell population is free of epithelial cells.

24. The method of claim 23, wherein the mixed cell population comprises a limbal stromal niche cell, an umbilical cord stem cell, an amniotic membrane stem cell or an adipose stem cell.