Trophoblast cell preparations

• US 6,630,349 B1
• Filed: 03/07/2001
• Issued: 10/07/2003
• Est. Priority Date: 09/23/1998
• Status: Expired due to Fees

• Current Assignee: Mount Sinai Hospital (Sinai Health System)
• Original Assignee: Mount Sinai Hospital (Sinai Health System)
• Inventors: Tanaka, Satoshi, Rossant, Janet, Kunath, Tilo
• Primary Examiner(s): Wehbe′ , Anne M.
• Assistant Examiner(s): LI, QIAN JANICE
• Application Number:
  US09/786,585
• Time in Patent Office: 944 Days
• Field of Search: 435/325, 435/455, 435/373, 435/377, 800/21
• US Class Current: 435/325
• CPC Class Codes: A61K 35/12 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 Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C12N 2501/119 Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
• CPC Class Codes: C12N 2501/91 Heparin
• CPC Class Codes: C12N 2502/13 connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
• CPC Class Codes: C12N 5/0605 Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly

Abstract

Stable pluripotent trophoblast stem (TS) cell lines and uses of the cell lines are described. The cell lines comprise cells that (i) are capable of indefinite proliferation in vitro in an undifferentiated state, and (ii) are capable of differentiation into cells of the trophoblast lineage in vivo.

Claims

1. An isolated preparation of mouse trophoblast stem cells which (i) are capable of indefinite proliferation in vitro in an undifferentiated state;

(ii) are capable of differentiation into cells of the trophoblast lineage in vivo, and (iii) are diploid trophoblast cells.

2. An isolated preparation as claimed in claim 1 wherein the cells are characterized by expression of the genetic markers Errβ

, Cdx2 and Fgfr2.

3. The isolated preparation according to claim 1 which is derived from or comprised of cells that have been genetically modified by genetic engineering techniques.

4. An isolated cell preparation as claimed in claim 3 modified by introducing mutations into genes in the cells or by introducing transgenes into the cells.

5. A method for producing a preparation of mouse trophoblast stem cells as claimed in claim 1 comprising culturing early postimplantation trophoblast cells or cells of a blastocyst from a mouse on a feeder laser in the presence of exogenous FGF4 and heparin, said feeder layer selected from the group consisting of fibroblasts or medium conditioned by fibroblasts.

6. A method for producing differentiated cells of trophoblast lineages comprising culturing early postimplantation trophoblast cells or cells of a blastocyst from a mouse as claimed in claim 5 and inducing differentiation of the stern cells to cells of trophoblast lineages by removing the FGF4, the heparin, or the feeder layer.

7. A method as claimed in claim 5 wherein the early postimplantation trophoblast cells or cells of a blastocyst are transgenic.

8. A method as claimed in claim 5 wherein the feeder layer is a confluent fibroblast layer or a medium conditioned by primary embryonic fibroblast cells.

9. A method as claimed in claim 5 wherein the feeder layer comprises primary mouse embryonic fibroblast (EMFI) cells or STO cells.

10. A method as claimed in claim 5 wherein the FGF4 is recombinant FGF4.

11. A method for producing chimeric conceptuses or placenta comprising the steps of producing a preparation of mouse trophoblast stem cells as claimed in claim 5 and further comprising the step of introducing cells frown the preparation into a non-human blastocyst or aggregating the cells with an early stage non-human embryo to produce chimeric conceptuses or placenta.

12. A method as claimed in claim 11 wherein the chimeric conceptuses or placenta are engineered to carry selectable markers or genetic alterations.

13. A method as claimed in claim 11 wherein cell lines are derived from the chimeric conceptuses or chimeric placenta.

14. A method for producing a preparation of mouse trophoblast stem cells as claimed in claim 1 comprising:

(a) isolating extraembryonic cells from embryos or blastocysts from a mouse;

(b) culturing the extraembryonic cells on a feeder layer in the presence of exogenous FGF4 and heparin to thereby obtain an isolated preparation of mouse trophoblast stem cells, said feeder layer selected from the group consisting of fibroblasts or medium conditioned by fibroblasts.

15. A method for producing differentiated cells of trophoblast lineages comprising culturing extraembryonic cells according to claim 14, and inducing differentiation of the stem cells to cells of trophoblast lineages by removing the FGF4, the heparin, or the feeder layer.

16. A method for screening for potential therapeutics that modulate trophoblast development or activity comprising subjecting an isolated preparation of mouse trophoblast stem cells which (i) are capable of indefinite proliferation in vitro in an undifferentiated state;

(ii) are capable of differentiation into cells of the trophoblast lineage in vivo, and (iii) are diploid trophoblast cells, to a test substance, and comparing the effect of the test substance to a control to determine if the test substance modulates trophoblast development or activity.