Differentiation of BECs from hESCs/iPSCs

BEC differentiation was adapted from Qian et al., 2017^{17 (link)}. Human ESC/iPSC’s were disassociated to single cell via Accutase and reseeded at 35*10³/cm² onto matrigel coated plates in mTeSR1 supplemented with 10 μM Y27632 (Stem Cell Technologies). For the next two days, media was replaced with mTesR1 medium daily. On the third day, the medium as changed to DeSR1 medium (DMEM/F12 with Glutamax (Life Technologies) Supplemented with 0.1 mM B-mercaptoethanol, 1X MEM-NEAA, 1X penicillin-streptomycin and 6 μM CHIR99021 (R&D Systems). The following 5 days the medium was changed to DeSR2 (DMEM/F12 with Glutamax (Life Technologies) Supplemented with 0.1 mM B-mercaptoethanol, 1X MEM-NEAA, 1X penicillin-streptomycin and B-27 (Invitrogen)) and changed every day. After 5 days of DeSR2, the medium was changed to hECSR1 Human Endothelial SFM (ThermoFisher) supplemented with B-27, 10 μM retinoic acid and 20 ng/mL bFGF. The BEC’s were then split using Accutase and reseeded with hECSR1 supplemented with 10 μM Y27632. The BECs were then maintained through hECSR2 medium (hECSR1 medium lacking RA+bFGF).

Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link: Access Free Full Text.

Blanchard J.W., Bula M., Davila-Velderrain J., Akay L.A., Zhu L., Frank A., Victor M.B., Bonner J.M., Mathys H., Lin Y.T., Ko T., Bennett D.A., Cam H.P., Kellis M, & Tsai L.H. (2020). Reconstruction of the Human Blood-Brain Barrier in vitro reveals a Pathogenic Mechanism of APOE4 in Pericytes. Nature medicine, 26(6), 952-963.

Publication 2020

Y27632 DMEM/F12 with Glutamax penicillin-streptomycin CHIR99021 Human Endothelial SFM

Accutase Cell Chir99021 Endothelial Human Ipsc Matrigel Mercaptoethanol Penicillin Retinoic acid Stem cell Streptomycin Y27632

Corresponding Organization :

Other organizations : Massachusetts Institute of Technology, Rush University Medical Center

Top 5 similar protocols

Protocol cited in 1 other protocol

Variable analysis

independent variables

Differentiation protocol (BEC differentiation adapted from Qian et al., 2017)

dependent variables

Differentiation of human ESC/iPSC into BECs

control variables

Cell seeding density (35*10^3/cm^2)
Culture substrate (Matrigel coated plates)
Initial culture medium (mTeSR1 supplemented with 10 μM Y27632)
Subsequent culture media (DeSR1, DeSR2, hECSR1, hECSR2)

positive controls

Not specified

negative controls

Not specified

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!