Blastoid, cell line based artificial blastocyst

Patent metadata

Field	Value
Publication number	US-9822336-B2
Application number	US-201414784659-A
Country	US
Kind code	B2
Filing date	Apr 16, 2014
Priority date	Apr 16, 2013
Publication date	Nov 21, 2017
Grant date	Nov 21, 2017

Abstract

A method for making an at least double layered cell aggregate and/or an artificial blastocyst, and/or a further-developed blastoid termed blastoid, by forming a double layered cell aggregate from at least one trophoblast cell and at least one pluripotent and/or totipotent cell, and culturing the aggregate to obtain an artificial blastocyst having a trophectoderm-like tissue that surrounds a blastocoel and an inner cell mass-like tissue. The cell aggregate can be formed from toti- or pluripotent stem cell types, or induced pluripotent stem cell types, in combination with trophoblast stem cells. Formation of a blastoid can be achieved by culturing the cell aggregate in a medium preferably comprising one or more of a Rho/ROCK inhibitor, a Wnt pathway modulator, a PKA pathway modulator, a PKC pathway modulator, a MAPK pathway modulator, a STAT pathway modulator, an Akt pathway modulator, a Tgf pathway modulator and a Hippo pathway modulator. Also, a method for growing an at least double layered cell aggregate into an artificial blastocyst, and into a further-developed blastoid, a foetus or a live animal and an in vitro cell culture comprising the mentioned compounds and/or cell aggregates.

First claim

The invention claimed is:

1. An in vitro method of making an at least double layered mouse cell aggregate, the method comprising:
seeding between 2 and 14 mouse embryonic stem (ES) cells into a flat-bottom microwell with a diameter of between 100 and 500 micrometers;
culturing the ES cells in a first culture medium to form an aggregate;
seeding between 3 and 20 mouse trophoblast stem (TS) cells into the microwell in a second culture medium; and
culturing the mixture of ES and TS cells to obtain an at least double layered cell aggregate.

2. The method according to claim 1 , wherein the embryonic stem cells and/or trophoblast stem cells are obtained from a cell line.

3. The method according to claim 1 , wherein the microwell has a non-adherent surface.

4. The method according to claim 1 , wherein the first and/or second culture medium comprises a Rho/ROCK inhibitor.

5. The method according to claim 1 , wherein the first and/or second culture medium comprises a modulator of a pathway selected from the group consisting of the Writ-pathway, the PKA pathway, the PKC pathway, the MAPK pathway, the STAT pathway, the Akt pathway, the Tgf pathway and the Hippo pathway.

6. The method according to claim 5 , wherein the modulator of the Wnt-pathway is an activator of the Wnt-pathway.

7. The method according to claim 6 , wherein the activator of the Wnt-pathway is a glycogen synthase kinase inhibitor or a Wnt agonist.

8. The method according to claim 5 , wherein the modulator of the PKA pathway is an activator of the PKA pathway.

9. An at least double layered cell aggregate obtained by the method according to claim 1 .

Assignees

• Univ Maastricht
• Koninklijke Nederlandse Akademie Van Wetenschappen

Inventors

• Rivron Nicolas Clément
• Van Blitterswijk Clemens Antoni
• Geijsen Niels
• Vrij Erik Jacob

Classifications

• C12N2535/00

  Supports or coatings for cell culture characterised by topography · CPC title

• C12N2517/00

  Cells related to new breeds of animals · CPC title

• C12N2502/025

  extra-embryonic cells, e.g. amniotic epithelium, placental cells, Wharton's jelly · CPC title

• C12N2502/02

  embryonic cells · CPC title

• C12N2501/91

  Heparin · CPC title