Differentiation of multi-lineage progenitor cells to respiratory epithelial cells
View Patent ↗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.
1. A method of producing a population of cells having a respiratory epithelial cell phenotype, said method comprising culturing a purified population of human fetal blood multi-lineage progenitor cells (MLPC) or a clonal line of human fetal blood MLPC with a differentiation medium effective to induce differentiation of said MLPC into cells having said respiratory epithelial cell phenotype, wherein said MLPC within said purified population or clonal line are positive for CD9, CD13, CD29, CD44, CD73, CD90 and CD105, and, negative for CD10, CD34, CD41, CD45, Stro-1, stage specific embryonic antigen (SSEA)-3, and SSEA-4.
2. The method of claim 1 , wherein said differentiation medium comprises hydrocortisone, epidermal growth factor, insulin, triiodothyronine, transferrin, bovine serum albumin, retinoic acid, pituitary extract, and epinephrine.
3. The method of claim 1 , further comprising testing said cells having said respiratory epithelial cell phenotype for surfactant protein C.
4. The method of claim 3 , wherein testing said cells having said respiratory epithelial cell phenotype comprises staining said cells with an antibody having binding affinity for prosurfactant protein C.
5. 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-CD 15 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; 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.
6. The method of claim 5 , said method further comprising testing said cells having said respiratory epithelial cell phenotype for surfactant protein C.
7. The method of claim 5 , said method further comprising producing a clonal line of MLPC from said MLPC having said fibroblast morphology before culturing with said differentiation medium.