STEM CELL RESEARCH

Gating strategy for LT-HSCs
Gating strategy for LT-HSCs
Frozen human cord blood mononuclear cells (StemCell Technologies) were enriched using the BD IMag™ human lineage cell depletion set – DM (Cat. No. 560030), stained with antibodies, and acquired and analyzed on a BD LSRFortessa™ flow cytometer. A. First, cells were gated based on light-scatter properties to screen out debris. B. Next, viable, lineage-negative (CD2, CD3, CD4, CD7, CD8, CD10, CD11b, CD14, CD19, CD20, CD56, CD235a) cells were gated based on the viability dye 7-AAD and the BD Pharmingen™ human lineage cocktail 4 kit (Cat. No. 562722). To identify a highly enriched LT-HSC population, (C) cells were gated on CD34+CD38; (D) then (in a child gate) on CD90+CD45RA; and (E) finally on CD49f+. This combination of cell surface markers, summarized in (F) the complete gating hierarchy, results in a population rich in LT-HSCs.
Changes in transcription factors in definitive endoderm development
Changes in transcription factors in definitive endoderm development
H9 hESCs (WiCell) were differentiated into definitive endoderm according to D’Amour, et al2 and monitored on a BD LSRFortessa™ flow cytometer using the BD Stemflow™ definitive and pancreatic endoderm analysis kit (Cat. No. 562496). As embryonic stem cells (A) differentiated toward definitive endoderm (B), more cells expressed both Sox17 and FoxA2, while Nanog expression decreased.
Changes in intracellular and surface markers in neural cell differentiation
Changes in intracellular and surface markers in neural cell differentiation
NSCs derived from H9 hESCs using the serum-free embryoid body (SFEB) method were differentiated into neurons and glia and monitored on a BD LSRFortessa flow cytometer using the BD Stemflow™ human neural lineage analysis kit (Cat. No. 561526). A. As differentiation progressed, cells expressed less Nestin and more DCX. B. Nestin+ cells were further delineated into a glial cell population that expressed both CD44 and Nestin over time.
Using surface marker screening to characterize and enrich neural stem cells and neurons
Using surface marker screening to characterize and enrich neural stem cells and neurons
A. H9 hESCs were induced using the SFEB method,1 and the BD Lyoplate™ human cell surface marker screening panel (Cat. No. 560747) was used to screen the resulting heterogeneous neural cultures on a BD™ LSR II flow cytometer to identify a surface marker signature for NSCs. After identifying potential hits, NSC cell surface phenotypes were verified and a CD184+CD44 CD271CD24+CD15mid sorting profile was used on a BD FACSAria™ II cell sorter to obtain a near-pure subpopulation of NSCs.
Using surface marker screening to characterize and enrich neural stem cells and neurons
Using surface marker screening to characterize and enrich neural stem cells and neurons
B. The purified NSCs were differentiated into a mixed culture of neuronal and glial cell populations, which was screened using the same panel to identify a surface marker signature for neurons. An imaging screen was chosen due to the unique morphology of neurons and the ability to co-stain with a neuron-specific marker. Potential hits were verified by flow cytometry, and a CD44 CD184CD24+CD15low sorting profile was used to purify neurons.1
Viability of H9 hESCs following sorting
Viability of H9 hESCs following sorting

In the experiment shown, pluripotent H9 embryonic stem cells (TRA-1-81+, SSEA-3+, SSEA-1-) were sorted from a cultured population using a new comprehensive flow cytometry kit from BD Biosciences, the BD Stemflow™ human pluripotent stem cell sorting and analysis kit (Cat. No. 560461) on a BD FACSAria™ II cell sorter.

A) The plot shows the scatter plot of the cells established using BD™ CompBead Plus microparticles. Populations are shown as clusters defined by two markers.

Viability of H9 hESCs following sorting
Viability of H9 hESCs following sorting

In the experiment shown, pluripotent H9 embryonic stem cells (TRA-1-81+, SSEA-3+, SSEA-1-) were sorted from a cultured population using a new comprehensive flow cytometry kit from BD Biosciences, the BD Stemflow™ human pluripotent stem cell sorting and analysis kit (Cat. No. 560461) on a BD FACSAria™ II cell sorter.

B) To demonstrate cell viability, pluripotent cells were cultured following sorting, brightfield imaged, and examined for karyotypic abnormalities. Sorted cells were viable and displayed a normal karyotype.

Viability of H9 hESCs following sorting
Viability of H9 hESCs following sorting

In the experiment shown, pluripotent H9 embryonic stem cells (TRA-1-81+, SSEA-3+, SSEA-1-) were sorted from a cultured population using a new comprehensive flow cytometry kit from BD Biosciences, the BD Stemflow™ human pluripotent stem cell sorting and analysis kit (Cat. No. 560461) on a BD FACSAria™ II cell sorter.

C) To demonstrate cell viability, pluripotent cells were cultured following sorting, brightfield imaged, and examined for karyotypic abnormalities. Sorted cells were viable and displayed a normal karyotype.

Pluripotency of H9 hESCs following sorting
Pluripotency of H9 hESCs following sorting

In the experiment shown, pluripotent H9 embryonic stem cells (TRA-1-81+, SSEA-3+, SSEA-1-) were sorted from a cultured population using the BD Stemflow™ Human Pluripotent Stem Cell Sorting and Analysis Kit (Cat. No. 560461) on a BD FACSAria™ II cell sorter.

A) Key markers for pluripotency (SSEA-4 Alexa Fluor® 647 (Cat. No. 560219) pseudocolored green, Oct3/4 Alexa Fluor® 555 (Cat. No. 560306) pseudocolored yellow, and SSEA-1 Alexa Fluor® 488 (Cat. No. 560172) pseudocolored red) were analyzed by imaging on a BD Pathway™ bioimager.

Pluripotency of H9 hESCs following sorting
Pluripotency of H9 hESCs following sorting

Sorted cells expressed markers for pluripotency. Undifferentiated Oct3/4+SSEA-4+, SSEA-4+SSEA-1 cells composed 93.8% and 95.3% of the cell population, respectively.

Bioimaging of hESCs cultured on BD Matrigel matrix
Flow cytometry analysis of hESCs cultured on BD Matrigel matrix

H9 hESCs were cultured in mTeSR™1 maintenance medium on BD Falcon™ 96-well imaging plates (Cat. No.353219) that were coated with BD Matrigel hESC-qualified matrix (Cat. No. 354277). Cells were fixed with 4% paraformaldehyde, followed by BD Perm/Wash™ buffer (Cat.No. 554723). Multicolor cell staining was performed by incubating cells with the following fluorochrome-conjugated antibodies: Sox2 Alexa Fluor® 647 (Cat. No. 560302) pseudocolored yellow, Oct3/4 Alexa Fluor® 555 (Cat. No. 560306) pseudocolored red, and SSEA-4 Alexa Fluor® 488 (Cat. No. 560308) pseudocolored green. Cell nuclei were counterstained using Hoechst 33342 pseudocolored blue. The cells were imaged on a BD Pathway™ 435 bioimager using a 10x objective.


References

1. Yuan SH, Martin J, Elia J, et al. Cell surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells. PLoS One. 2011;6:e17540.

2. D’Amour KA, Agulnick AD, Eliazer S, Kelly OG, Kroon E, Baetge EE. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol. 2005;23:1534-1541.