Granted Patent
B2
US 12,467,917 · App. 17/692,790 · Granted Nov 11, 2025
Reconstructing human early embryogenesis in vitro with pluripotent stem cells
Inventors:
Magdalena D. Zernicka-Goetz (Pasadena, CA); Berna Sozen (Pasadena, CA); Victoria Jorgensen (Pasadena, CA)
Assignee:
California Institute of Technology
G01N33/5005C12N5/0604C12N5/0606C12N5/0697C12N2310/14C12N2501/113C12N2501/115C12N2501/15C12N2501/155C12N2501/415C12N2503/04C12N2506/45C12N2513/00
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Abstract
Disclosed herein include methods and compositions for in vitro culture of three-dimensional expanded pluripotency (EP) structures from pluripotent stem cells. In some embodiments, the method can include generating expanded pluripotent stem cells (EPSCs) and culturing the EPSCs in a composition capable of supporting generation of the EP structure.
Claims (27)
1 . An in vitro method of generating an expanded pluripotency (EP) structure in three dimensions, the method comprising:
(a) contacting pluripotent stem cells (PSCs) with EP media and culturing the PSCs in the EP media to generate expanded pluripotent stem cells (EPSCs);
(b) contacting at least 5 EPSCs with a first substrate and a composition capable of supporting generation of an EP structure, wherein the composition comprises 20% to 30% EP media, 20% to 30% trophoblast stem cell (TSC) media, 45% to 55% in vitro fertilization (IVF) media, and an ALK5 kinase inhibitor, and wherein the composition further comprises FGF2 to facilitate cavitation of the EP structure and one or more of: a TGFβ ligand, a WNT agonist, and a ROCK inhibitor; and
(c) culturing the EPSCs in the composition for at least 4 days, and wherein step (c) comprises reducing the concentration of FGF2 in the composition by half and removing the ALK5 kinase inhibitor after at least 48 hours of culturing in the composition, wherein the EPSCs self-organize to generate the EP structure, wherein the EP structure comprises a single outside layer, an enlarged cavity, and an internal acentric compartment.
2 . The in vitro method of claim 1 , wherein the composition comprises 15 ng/ml to 25 ng/ml BMP4, 1.5 μM to 2.5 μM CHIR99021, 35 ng/ml to 45 ng/ml FGF2, 4.5 μM to 6 μM Y-27632, and 1.5 μM to 2.5 μM A83-01.
3 . The in vitro method of claim 1 , wherein the PSCs are induced pluripotent stem cells (iPSCs) or mammalian embryonic stem cells (ESCs).
4 . The in vitro method of claim 3 , wherein the PSCs, iPSCs, or ESCs are genetically modified.
5 . The in vitro method of claim 1 , wherein the EPSCs form dome-shaped colonies, and wherein step (b) comprises isolating single EPSCs by contacting the dome-shaped colonies with a cell detachment enzyme and resuspending the isolated single EPSCs in EP media.
6 . The in vitro method of claim 1 , wherein the first substrate comprises a dish, a U-plate, a flask, or a microwell plate, and wherein the microwell plate comprises inverted pyramidal microwells.
7 . The in vitro method of claim 1 , wherein the EP media comprises a basal media, N2 supplement, 0.1% to 2% non-essential amino acids, 0.01 mM to 0.2 mM beta-mercaptoethanol (BME), Penicillin-streptomycin, 5 ng/ml to 15 ng/ml leukemia inhibitory factor (LIF), 0.5 mM to 1.5 mM CHIR99021, 0.5 mM to 1.5 mM S-(+)-Dimethindenemaleate, and/or 1.5 mM to 2.5 mM Minocycline HCl.
8 . The in vitro method of claim 1 , wherein the TSC media comprises a basal media, 0.01 mM to 0.2 mM BME, 0.1% to 1% fetal bovine serum (FBS), 0.1% to 1% Penicillin-streptomycin, 0.1% to 1% bovine serum albumin (BSA), 0.1% to 2% ITS-X, 1 ng/ml to 2 ng/ml ascorbic acid, 45 ng/ml to 55 ng/ml EGF, 1.5 mM to 2.5 mM CHIR99021, 0.4 mM to 1 mM A83-01, 0.5 mM to 1.5 mM SB431542, 0.5 mM to 1.5 mM valproic acid (VPA), and 4.5 mM to 6 mM Y-27632.
9 . The in vitro method of claim 1 , wherein the IVF media comprises a continuous culture media.
10 . The in vitro method of claim 1 , wherein step (c) comprises culturing the EPSCs in hypoxic conditions, and wherein the hypoxic conditions comprise 5% oxygen.
11 . The in vitro method of claim 1 , wherein the single outside layer of the EP structure comprises one or more cells expressing KRT18, GATA3, or both, and wherein the internal acentric compartment of the EP structure comprises one or more cells expressing SOX2, SOX17, OCT4, FOXA2, or any combination thereof.
12 . The in vitro method of claim 1 , wherein the EP structure comprises 60 to 260 cells and/or wherein the EP structure is 100 μm to 200 μm in diameter.
13 . The in vitro method of claim 1 , wherein expression of one or more trophectoderm marker genes, one or more epiblast marker genes, one or more hypoblast marker genes, or any combination thereof, are increased in the EP structure by at least 2-fold following at least four days of culture in the composition relative to the EPSCs generated in step (a), wherein the one or more trophectoderm marker genes comprise GATA3, PLAC8, CDX2, KRT8, KRT18, or any combination thereof, wherein the one or more epiblast marker genes comprise KLF4, and wherein the one or more hypoblast marker genes comprise PDGFRA, GATA6, or both.
14 . The in vitro method of claim 1 , further comprising:
(d) contacting the EP structure with a second substrate and in vitro culture (IVC) media, wherein the IVC media comprises:
(i) insulin, an insulin analogue, or an insulin receptor agonist;
(ii) estrogen, an estrogen analogue, or an estrogen receptor agonist; and
(iii) progesterone, a progesterone analogue, or a progesterone receptor agonist; and
(e) culturing the EP structure in the IVC media for at least 24 hours, wherein the EP structure reorganizes to form a reorganized EP structure comprising an outer compartment, an inner compartment, and an internal lumen.
15 . The in vitro method of claim 14 , wherein the second substrate comprises a dish, a U-plate, a flask, or a microwell plate.
16 . The in vitro method of claim 14 , wherein the outer compartment comprises one or more cells expressing one or more extra-embryonic markers, and the inner compartment comprises one or more cells expressing one or more embryonic markers, and wherein the one or more extra-embryonic-markers comprise GATA3, KRT18, FOXA2, TFAP2, or any combination thereof and the one or more embryonic markers comprise SOX2.
17 . The in vitro method of claim 1 , further comprising:
removing one or more cells from the EP structure and culturing the one or more cells to produce differentiated cells.
18 . The in vitro method of claim 1 , wherein the ALK5 kinase inhibitor is A83-01.
Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded May 18, 2022
From: ZERNICKA-GOETZ, MAGDALENA; SOZEN, BERNA; JORGENSEN, VICTORIA
To: CALIFORNIA INSTITUTE OF TECHNOLOGY
Reel/Frame 059951/0528 →
Continuity (2)
Provisional Application
63161935
· Mar 16, 2021
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