Generation of matrix-bound nanovesicles from human brain organoids
AssigneeThe Florida State University Research Foundation, Inc.
InventorsCHANG LIU, YAN LI
Life of this patent
Estimating the term…
Abstract
The subject invention pertains to novel methods of isolating and differentiating extracellular vesicles (EVs) from a subject, particularly from human induced pluripotent stem cells (hiPSCs). The EVs can be isolated from the spent media of 3D brain organoids differentiated from hiPSCs or from the decellularized extracellular matrix (ECM) of brain organoids.
Claims19
A method for preparing a media extracellular vesicle (MEV) or a matrix-bound nanovesicles (MBV), said method comprising:
- i) providing a human induced pluripotent stem cell (hiPSC);
- ii) incubating the hiPSC with a protein kinase inhibitor for about 1 day;
- iii) incubating the hiPSC with an activin receptor-like kinase receptor inhibitor for about 7 days;
- iv) incubating the hiPSC with a growth factor protein for about 7 days to yield a hiPSC-derived forebrain cortical organoid (iFCo);
- v) harvesting the iFCo;
- and
- vi) isolating the MEV from the iFCo or isolating the MBV from the iFCo.
A method for preparing a media extracellular vesicle (MEV), said method comprising:
- i) providing a human induced pluripotent stem cell (hiPSC);
- ii) incubating the hiPSC with a protein kinase inhibitor and an activin receptor-like kinase receptor inhibitor for about 7 days;
- iii) incubating the hiPSC with a first growth factor protein for about 5 days;
- iv) incubating the hiPSC with retinoic acid and a glycogen synthase kinase-3 inhibitor for about 7 days;
- v) incubating the hiPSC with a second growth factor protein for about 7 days;
- vi) removing the second growth factor protein and incubating the hiPSC for about 7 days;
- vii) incubating the hiPSC with an alpha chemokine and a hedgehog pathway activator for about 7 days to yield a hiPSC-derived hindbrain cerebellar organoid (iHCo);
- viii) harvesting the iHCo;
- and
- ix) isolating the MEV from the iHCo.
A method for preparing a MEV or a matrix-bound nanovesicle MBV, said method comprising:
- i) providing a human induced pluripotent stem cell (hiPSC);
- ii) incubating the hiPSC with a protein kinase inhibitor and an activin receptor-like kinase receptor inhibitor for about 7 days;
- iii) incubating the hiPSC with a growth factor protein for about 5 days;
- iv) incubating the hiPSC with retinoic acid and a glycogen synthase kinase-3 inhibitor for about 7 days to yield a hiPSC-derived hindbrain cerebellar organoid (iHCo);
- v) harvesting the iHCo;
- and
- vi) isolating the MEV from the iHCo or isolating the MBV from the iHCo.
Dependencies are inferred from the claim text.