Differentiation of Multi-Lineage Progenitor Cells to Respiratory Epithelial Cells

• US 20070249047A1
• Filed: 04/17/2007
• Published: 10/25/2007
• Est. Priority Date: 04/17/2006
• Status: Active Grant

• Current Assignee: BioE LLC, Regents of The University of Minnesota (University of Minnesota)
• Original Assignee: BioE, Inc., Regents of The University of Minnesota (University of Minnesota)
• Inventors: Berger, Michael, McKenna, David, Tigges, Barbara
• Granted Patent: US 7,727,763 B2
• Time in Patent Office: Days
• US Class Current: 435/372
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/84 from mammals
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/395 Thyroid hormones
• CPC Class Codes: C12N 2501/81 Adrenaline
• CPC Class Codes: C12N 2506/03 from non-embryonic pluripotent stem cells
• CPC Class Codes: C12N 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0688 Cells from the lungs or the respiratory tract

Abstract

Fetal blood multi-lineage progenitor cells that are capable of a wide spectrum of transdifferentiation are described, as well as methods of differentiating the progenitor cells into type II alveolar cells.

Claims

1. A composition comprising a purified population of human fetal blood multi-lineage progenitor cells (MLPC) or a clonal line of human fetal blood MLPC and a differentiation medium effective to induce differentiation of said MLPC into cells having a respiratory epithelial cell phenotype, wherein said MLPC are positive for CD9, negative for CD45, negative for CD34, and negative for SSEA-4.

2. The composition of claim 1, wherein said differentiation medium comprises hydrocortisone, epidermal growth factor, insulin, triiodothyronine, transferrin, and bovine serum albumin.

3. The composition of claim 2, said differentiation medium further comprising retinoic acid, pituitary extract, and epinephrine.

4. The composition of claim 2, said differentiation medium further comprising an antibiotic.

5. The composition of claim 1, said composition further comprising an antibody with binding affinity for prosurfactant protein C.

6. A composition comprising a mixture of cells, said mixture comprising MLPC and cells having a respiratory epithelial cell phenotype.

7. The composition of claim 6, said composition further comprising a culture medium or a differentiation medium.

8. The composition of claim 7, wherein said differentiation medium comprises hydrocortisone, epidermal growth factor, insulin, triiodothyronine, transferrin, bovine serum albumin, retinoic acid, pituitary extract, and epinephrine.

9. The composition of claim 7, wherein said culture medium or said differentiation medium comprises a cryopreserative.

10. A method of producing a population of cells having a respiratory epithelial cell phenotype, said method comprising culturing a purified population of MLPC or a clonal line of MLPC with a differentiation medium effective to induce differentiation of said MLPC into cells having said respiratory epithelial cell phenotype, wherein said MLPC are positive for CD9, negative for CD45, negative for CD34, and negative for SSEA-4.

11. The method of claim 10, wherein said differentiation medium comprises hydrocortisone, epidermal growth factor, insulin, triiodothyronine, transferrin, and bovine serum albumin.

12. The method of claim 11, wherein said differentiation medium further comprises retinoic acid, pituitary extract, and epinephrine.

13. The method of claim 10, further comprising testing said cells having said respiratory epithelial cell phenotype for surfactant protein C.

14. The method of claim 13, wherein said cells having said respiratory epithelial cell phenotype are stained with an antibody having binding affinity for prosurfactant protein C.

15. A method for producing a population of cells having a respiratory epithelial cell phenotype from human fetal blood, said method comprising:

a) contacting a human fetal blood sample with a composition, said composition comprising;

i) dextran;

ii) anti-glycophorin A antibody;

iii) anti-CD15 antibody; and

iv) anti-CD9 antibody;

b) allowing said sample to partition into an agglutinate and a supernatant phase;

c) recovering cells from said supernatant phase;

d) purifying MLPC from the recovered cells by adherence to a solid substrate, wherein said MLPC are positive for CD9 and positive for CD45;

e) culturing said MLPC such that said MLPC obtain a fibroblast morphology; and

f) culturing said MLPC having said fibroblast morphology with a differentiation medium effective to induce differentiation of said MLPC into cells having said respiratory epithelial cell phenotype.

16. The method of claim 15, said method further comprising testing said cells having said respiratory epithelial cell phenotype for surfactant protein C.

17. The method of claim 15, said method further comprising producing a clonal line of MLPC from said MLPC having said fibroblast morphology before culturing with said differentiation medium.

18. A clonal population of cells having a respiratory epithelial cell phenotype.

19. The clonal population of claim 18, wherein said cells have enhanced expression of mRNA for a lysosomal ATPase relative to a clonal population of MLPC.

20. A composition comprising the clonal population of cells of claim 18 and a culture medium.

21. The composition of claim 20, said composition further comprising a cryopreservative.

22. The composition of claim 21, wherein said cryopreservative is dimethylsulfoxide (DMSO).

23. The composition of claim 22, wherein said cryopreservative is 1 to 10% DMSO.

24. The composition of claim 21, wherein said cryopreservative is fetal bovine serum, human serum, or human serum albumin in combination with one or more of the following:

DMSO, trehalose, and dextran.

25. The composition of claim 24, wherein said cryopreservative is human serum, DMSO, and trehalose.

26. The composition of claim 24, wherein said cryopreservative is fetal bovine serum and DMSO.

27. An article of manufacture comprising the clonal population of cells of claim 18.

28. The article of manufacture of claim 27, wherein said clonal population is housed within a container.

29. The article of manufacture of claim 27, wherein said container is a vial or a bag.

30. The article of manufacture of claim 27, wherein said container further comprises a cryopreservative.