Exploring the effects of cell seeding density on the differentiation of human pluripotent stem cells to brain microvascular endothelial cells

Fluids and Barriers of the CNS

Eric V Shusta¹  Sean P Palecek¹  Michael K Hjortness¹  Scott G Canfield¹  Hannah K Wilson¹ 

[1] Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison 53706, WI, USA

关键词: Seeding density;    Tight junctions;    Efflux transporters;    Transendothelial electrical resistance;    Brain microvascular endothelial cells;    Human pluripotent stem cells (hPSCs);    In vitro human blood–brain barrier (BBB) model;   

Others  :  1201039
DOI  :  10.1186/s12987-015-0007-9

 received in 2015-03-05, accepted in 2015-04-24,  发布年份 2015

【 摘 要 】

Background

Brain microvascular-like endothelial cells (BMECs) derived from human pluripotent stem cells (hPSCs) have significant promise as tools for drug screening and studying the structure and function of the BBB in health and disease. The density of hPSCs is a key factor in regulating cell fate and yield during differentiation. Prior reports of hPSC differentiation to BMECs have seeded hPSCs in aggregates, leading to non-uniform cell densities that may result in differentiation heterogeneity. Here we report a singularized-cell seeding approach compatible with hPSC-derived BMEC differentiation protocols and evaluate the effects of initial hPSC seeding density on the subsequent differentiation, yield, and blood–brain barrier (BBB) phenotype.

Methods

A range of densities of hPSCs was seeded and differentiated, with the resultant endothelial cell yield quantified via VE-cadherin flow cytometry. Barrier phenotype of purified hPSC-derived BMECs was measured via transendothelial electrical resistance (TEER), and purification protocols were subsequently optimized to maximize TEER. Expression of characteristic vascular markers, tight junction proteins, and transporters was confirmed by immunocytochemistry and quantified by flow cytometry. P-glycoprotein and MRP-family transporter activity was assessed by intracellular accumulation assay.

Results

The initial hPSC seeding density of approximately 30,000 cells/cm² served to maximize the yield of VE-cadherin+ BMECs per input hPSC. BMECs displayed the highest TEER (>2,000 Ω × cm²) within this same range of initial seeding densities, although optimization of the BMEC purification method could minimize the seeding density dependence for some lines. Localization and expression levels of tight junction proteins as well as efflux transporter activity were largely independent of hPSC seeding density. Finally, the utility of the singularized-cell seeding approach was demonstrated by scaling the differentiation and purification process down from 6-well to 96-well culture without impacting BBB phenotype.

Conclusions

Given the yield and barrier dependence on initial seeding density, the singularized-cell seeding approach reported here should enhance the reproducibility and scalability of hPSC-derived BBB models, particularly for the application to new pluripotent stem cell lines.

【 授权许可】

   
2015 Wilson et al.

【 图 表 】

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