A microparticle approach to morphogen delivery within pluripotent stem cell aggregates
DOI: 10.1016/j.biomaterials.2013.05.079
Authors: Andrés M. Bratt-Leal; Anh H. Nguyen; Katy A. Hammersmith; Ankur Singh; Todd C. McDevitt
Step 1: iPSC/ESC maintainance
day 0 ~ 1
culture “adherent” mouse ESC in “N2B27” for “1” days. For ECM, use “Gelatin” at “0.5” “%”. Medium should contain factors shown in the following table, and should be changed once in “1” days.
| concentration | unit | |
|---|---|---|
| N2 | — | — |
| B27 | — | — |
| BSA | 0.05 | g/l |
| PSA | — | — |
| streptomycin | — | — |
| amphotericin | — | — |
| Glutamine | 0.002 | mol/l |
Ideally, “ESC” should be obtained at the end of culture.
Step 2: Primitive streak 1
day 1 ~ 2
culture “spheroid” mouse ESC in “ESGRO complete basal medium” for “1” days. Medium should contain factors shown in the following table, and should be changed once in “1” days.
| concentration | unit | |
|---|---|---|
| BMP4 | 1.0e-5 | g/l |
Step 3: mesoderm 1
day 2 ~ 4
culture “spheroid” mouse ESC in “ESGRO complete basal medium” for “2” days. Medium should contain factors shown in the following table, and should be changed once in “2” days.
| concentration | unit | |
|---|---|---|
| BMP4 | 1.0e-5 | g/l |
Ideally, “mesoderm” should express “Brachyury-T”, “KDR”, “Pax6”, “POU5F1” at the end of culture.