Monitoring certain proteins secreted in human pluripotent stem cell (hPSC) culture provides a fast, non-invasive way to identify changes in human stem cells’ pluripotency, thus overcoming a major hurdle in their development into scalable, reproducible, commercial applications.

Researchers in the United Kingdom have identified the proteomic and transcriptomic changes that indicate the early loss of pluripotency. The biomarkers could be detected within the first 48 hours after pluripotency dissolution began, thus providing an early warning system for imminent loss of pluripotency in hPSC cultures.

Among current methods, immunofluorescence detects pluripotency losses in individual cells rather than in entire cultures; flow cytometry has variable results; RNA-based assays are time-consuming and sacrifice pluripotent cells; and cell morphology is subjective.

In a recent paper, Susan J. Kimber, PhD, professor of stem and developmental biology at the University of Manchester, and colleagues reported that the “multiplexing of four E8- against four E6-enriched secretome biomarkers provides a robust, diagnostic metric for the pluripotent state.” [As used here, “secretome” refers to all the proteins in the conditioned media.]

They removed the FGF-2 and TFGß growth factors from the E6-conditioned medium to cause the cells to lose their pluripotency, which they noted is “a prerequisite for lineage differentiation.” Then they analyzed the culture medium to identify protein biomarkers as the cells’ ability to differentiate waned, but while differentiation was still possible.

The scientists identified 117 secreted proteins whose numbers significantly increased or decreased once the growth factors were removed from culture. From that list, eight were chosen for investigation.

Abundance ratios are key

Importantly, they determined that the relative abundance ratios of these proteins, rather than the increase or decrease of any set of biomarkers, was a more reliable metric to indicate waning pluripotency.

In particular, the ratios of Cochlin, FGF Receptor-1, Follistatin, and Olfactomedin-like 3 proteins increased when cells were cultured in E6 medium. “These were, therefore, indicators that hPSCs were losing pluripotency,” Kimber told GEN.

Conversely, the proteins that were highest in relative abundance when cells were cultured in the E8 medium were Chromogranin A, Nidogen 1, Neuronal Pentraxin-2, and Semaphorin 3A. “These decreased quickly as cells transitioned to differentiation, either using growth factor removal or a directed differentiation protocol and so appear to be biomarkers of robust pluripotent cell cultures,” Kimber said.

Additionally, the scientists noted a “significant positive correlation between the LogFC of significantly changed secreted proteins and RNA transcripts.”

Kimber and colleagues indicate this analysis method overcomes issues caused by cell seeding, changes in cell viability and health, and medium volume.

Returning the E6 cultures to E8 [pluripotency maintenance] medium “led to a full recovery of pluripotency,” they reported, enabling the cells to form teratomas. Full recovery took about seven days.

“The biomarkers would be useful for in-line monitoring of pluripotent stem cells in a commercial scale up setting, either as the starting material for drug discovery or stem cell-based therapy,” Kimber said. “However, they would also be useful in the research lab with a monitoring device.”

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