Differentiation of Multi-Lineage Progenitor Cells to Pancreatic Cells

• US 20090291494A1
• Filed: 05/20/2009
• Published: 11/26/2009
• Est. Priority Date: 05/21/2008
• Status: Abandoned Application

• Current Assignee: BioE LLC
• Original Assignee: BioE, Inc.
• Inventors: Collins, Daniel P.
• Application Number:
  US12/469,471
• Publication Number:
  US 20090291494A1
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/12 Hepatocyte growth factor [HGF]
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• 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 5/067 Hepatocytes
• CPC Class Codes: C12N 5/0676 Pancreatic cells

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 pancreatic cells.

Claims

1. A method of producing a population of cells having a pancreatic cell phenotype, said method comprising:

a) providing a collagen-coated culturing device housing a purified population of human fetal blood multi-lineage progenitor cells (MLPC) or a clonal line of MLPC, wherein said MLPC are positive for CD9, CD13, CD29, CD44, CD73, CD90, and CD105, and negative for CD10, CD34, CD41, CD45, Stro-1, SSEA-3, and SSEA-4;

b) culturing said purified population of MLPC or said clonal line of MLPC with a differentiation medium until cells having a hepatocytye-pancreatic precursor phenotype are obtained, said differentiation medium comprising ascorbic acid, hydrocortisone, transferrin, insulin, epidermal growth factor, hepatocyte growth factor, and stem cell factor; and

c) further culturing said cells having said hepatocytye-pancreatic precursor phenotype in said differentiation medium in the presence of retinoic acid to obtain cells having said pancreatic cell phenotype,.

2. The method of claim 1, further comprising testing said cells having said pancreatic cell phenotype for insulin, proinsulin, glucagon, PDX-1, or SOX-17.

3. The method of claim 2, said method further comprising testing said cells having said pancreatic cell phenotype for intracellular albumin.

4. A method for producing a population of cells having a pancreatic 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, wherein said MLPC having said fibroblast morphology are positive for CD9, CD13, CD29, CD44, CD73, CD90, and CD105, and negative for CD10, CD34, CD41, CD45, Stro-1, SSEA-3, and SSEA-4;

f) culturing said MLPC having said fibroblast morphology with a differentiation medium effective to induce differentiation of said MLPC into cells having a hepatocyte-pancreatic precursor cell phenotype, said differentiation medium comprising ascorbic acid, hydrocortisone, transferrin, insulin, epidermal growth factor, hepatocyte growth factor, and stem cell factor; and

g) culturing said cells having a hepatocyte-pancreatic precursor cell phenotype in said differentiation medium in the presence of retinoic acid to obtain cells having said pancreatic cell phenotype.

5. The method of claim 4, said method further comprising testing said cells having said pancreatic cell phenotype for insulin, proinsulin, glucagon, PDX-1, or SOX-17.

6. The method of claim 4, said method further comprising producing a clonal line of MLPC from said MLPC having said fibroblast morphology before culturing with said differentiation medium.

7. A clonal population of cells having a pancreatic cell phenotype.

8. A composition comprising the clonal population of cells of claim 7 and a culture medium.

9. The composition of claim 8, said composition further comprising a cryopreservative.

10. The composition of claim 9, wherein said cryopreservative is dimethylsulfoxide (DMSO).

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

12. The composition of claim 9, 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.

13. The composition of claim 12, wherein said cryopreservative is human serum, DMSO, and trehalose.

14. The composition of claim 12, wherein said cryopreservative is fetal bovine serum and DMSO.

15. An article of manufacture comprising the clonal population of cells of claim 7.

16. The article of manufacture of claim 15, wherein said clonal population is housed within a container.

17. The article of manufacture of claim 16, wherein said container is a vial or a bag.

18. The article of manufacture of claim 16, wherein said container further comprises a cryopreservative.

19. A composition comprising a purified population of 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 pancreatic cell phenotype, wherein said MLPC are positive for CD9, CD13, CD29, CD44, CD73, CD90, and CD105, and negative for CD10, CD34, CD41, CD45, Stro-1, SSEA-3, and SSEA-4.

20. The composition of claim 19, wherein said differentiation medium comprises ascorbic acid, hydrocortisone, transferrin, insulin, epidermal growth factor, hepatocyte growth factor, stem cell factor, and retinoic acid.

21. The composition of claim 19, said composition further comprising an antibody with binding affinity for insulin, proinsulin, glucagon, SOX-17, or PDX-1.