Detailed method description for noninvasive monitoring of differentiation status of human embryonic stem cells

Abstract

The (non)differentiation status of human embryonic stem cells (hESCs) is usually analyzed by determination of key pluripotency defining markers (e.g., OCT4, Nanog, SOX2) by means of reverse transcription quantitative polymerase chain reaction (RT-qPCR), flow cytometry (FC), and immunostaining. Despite proven usefulness of these techniques, their destructive nature makes it impossible to follow up on the same hESC colonies for several days, leading to a loss of information. In 2003, an OCT4-eGFP knock-in hESC line to monitor OCT4 expression was developed and commercialized. However, to the best of our knowledge, the use of fluorescence microscopy (FM) for monitoring the OCT4-eGFP expression of these cells without sacrificing them has not been described to date. Here, we describe such a method in detail, emphasizing both its resolving power and its complementary nature to FC as well as the potential pitfalls in standardizing the output of the FM measurements. The potential of the method is demonstrated by comparison of hESCs cultured in several conditions, both feeder free (vitronectin, VN) and grown on feeder cells (mouse embryonic fibroblasts, MEFs).

Assessing the impact of minimizing arginine conversion in fully defined SILAC culture medium in human embryonic stem cells

“…Because hESC are known to start differentiating spontaneously upon changes to their culture environment, the different conditions ( l‐ proline/ l‐ ornithine/Nor‐NOHA acetate/ l‐ arginine) were first analyzed for their effect on the differentiation status of the hESC by means of an optimized screening method using FM and FC (Fig. ) . A low eGFP expression (fluorescence signal <10 1 ), correlating to a low OCT4 expression, indicates differentiation.…”

“…In such experiments, the overall fluorescence of the colonies increases with time when no differentiation is induced, as we described earlier (Fig. A) . Normal E8 TM medium was used as a control.…”

“…Daily monitoring of OCT4 expression was assessed by noninvasive monitoring by means of FM. To assess influence of stress, a plate without daily monitoring (FM) but with FC at the end of the experiment was investigated to obtain information about OCT4 expression/cell and cell number as described earlier [8].…”

“…As stem cells are extremely sensitive to changes in their surroundings, we monitor pluripotency and general proteome changes following the different treatments intended to minimize conversion of heavy arginine. For this, an OCT4–eGFP Knock‐In hESC line (WiCell Research Institute, Madison, WI, USA) was used, and pluripotency was monitored by means of flow cytometry (FC) and fluorescence microscopy (FM) . To reduce the conversion, a completely defined culture for SILAC was supplemented by, respectively, adding different concentrations of l ‐proline, l ‐ornithine, or Nor‐NOHA acetate to the E8 culture medium, or by lowering the l ‐arginine concentration in this medium.…”

Embryonic Stem Cells: Keeping Track of the Pluripotent Status

“…Nevertheless, for those methods, ESC need to be harvested, which thus makes it impossible to monitor the same colony over and over again on a daily basis, as new samples have to be collected for each analysis. We present a method enabling pluripotency monitoring of the same specific colonies during a time-lapse experiment by using a reporter ESC line, as was published by Scheerlinck et al [55].…”