Over the last few weeks we’ve discussed the generation of induced pluripotent stem cells (iPSCs) with Sendai virus and Episomal Vectors and the subsequent expansion in feeder-free conditions with Essential 8™ Medium.

In this post we’ll discuss the identification of iPSC colonies during the process of somatic cell reprogramming.

Identification of true pluripotent colonies in a bed of transformed fibroblasts and other cell debris may be relatively easy for a trained eye, but challenging in the absence of extensive experience. Commonly, surface antibodies against pluripotent specific markers such as SSEA4, Tra-1-60 and Tra-1-81 are used but this method is expensive. Another option is the use of dyes that are substrates for markers of differentiation such as Alkaline Phosphatase (AP) and ABCG2 transporter. Exploiting markers of differentiation provides a way to monitor the progression of the reprogramming process.

Traditional AP substrates are harsh on the cells and once stained the cells cannot be propagated further. In contrast, the Molecular Probes® Alkaline Phosphatase Live Stain (AP Live) easily permeates into cells and once catalyzed results in a nontoxic byproduct that is easily diffused out of the cells.

How does Alkaline Phosphatase Live Stain work? 
The dye is a cell-permeable fluorescent substrate for alkaline phosphatase (AP) that is non-toxic to cells, diffusing out over the course of two hours. Simply wash cells in basal medium, dilute the dye, apply to cells, gently wash, and the cells are ready for fluorescent imaging. The diagram below outlines the protocol for AP Live use.

file

In addition, the following video describes the three critical steps required for success when using AP Live:


How do I visualize the stained cells?

After 15-20 minutes, remove the AP Live Stain and wash the cells three times for 5 minutes each with DMEM/F-12. Following the final wash, add fresh DMEM/F-12 and visualize the stained colonies under fluorescent microscopy using a standard FITC filter. Images should be captured within 30 minutes following the removal of the dye and the most robust fluorescent colonies should be marked for selection and expansion. Since the fluorescent signal leeches out of the cells and into the surrounding medium as the stain is turned over, we strongly encourage that you visualize and capture images immediately following the final wash for optimal signal detection.

How should cells stained with AP Live look?
The images below show various cells stained with the AP Live Stain. The differential staining of pluripotent stem cells easily distinguishes them from the feeder cells.

file


For further information, check out these two sources:

  • The open access publication by Singh et al. provides extensive information on the use of the Molecular Probes® Alkaline Phosphatase Live Stain during iPSC generation.
  • In the following video, Dr. Uma Lakshmipathy, the lead R&D scientist for AP Live, discusses the technology which allows for live staining of AP: