To ensure reproducible and consistent results across laboratories and to tackle possible safety concerns with stem cell-based therapies early in development, Creative Bioarray offers clients a variety of methods for assessing the pluripotency potential of iPSC and fully ensuring their characterization for registration and banking. In addition, we can provide customized solutions to meet the project requirements of our clients.
Background
Embryonic and pluripotent stem cells have the ability to differentiate into all cell lineages and remain undifferentiated during in vitro culture. Unlike ESCs derived from cell clusters within fertilized blastocysts, iPSCs can be induced directly from the patient's own somatic cells, thus circumventing the ethical issues associated with the destruction of embryos while greatly addressing potential immune rejection issues. These properties make iPSCs show significant promise for cell therapy applications.
Currently, the critical issue in clinical applications is to distinguish iPSCs from other cell types to avoid potential risks. Although morphological analysis has been used for their isolation, other more accurate isolation strategies are needed. Isolated iPSC clones differ in pluripotency markers expression and clones with similar levels of pluripotency marker expression exhibit different abilities in terms of lineage differentiation. Therefore, analysis of pluripotency marker would be a promising strategy for the isolation of iPSCs.
Fig.1 AP live staining assay (a) and in vivo detection of Tra-1-60 PE (red) (b) and Tra-1-60 FITC (green) (c) in an hESC line. (Martí, 2013)
Determining the Pluripotency of iPSCs
Creative Bioarray offers clients several methods for determining the pluripotency potential of iPSCs, including the detection of alkaline phosphatases (AP) associated with pluripotency and the assessment of various cellular markers by immunostaining techniques.
- AP expression
- iPSC maker expression
- TaqMan hiPSC scorecard assay
- qPCR and RNA-seq
AP is a hydrolase with high activity in pluripotent cells. The expression of AP can be easily determined by enzymatically converting the soluble colorimetric reagent to a precipitated state to produce a rapid visual readout, a rapid strategy for screening pluripotent cells using AP activity.
Detection of a set of markers specific to iPSC physiology by immunostaining techniques is another method used to confirm pluripotency. In humans, these markers typically include Oct4, Nanog, Sox2, Tra-1-60, Tra-1-81, SSEA3 and SSEA4. In mice, the major markers of pluripotency are Oct4, Sox2, Nanog, and SSEA1. Our researchers will provide a careful interpretation of such immunostaining data.
Advantages
- Mature immunofluorescence technologies
- High-efficient and reliable assays
- Customer-focused service and competitive pricing
Creative Bioarray is an experienced and highly specialized iPSC characterization service provider. Based on our advanced experimental platform and proven technology, we provide efficient iPSC pluripotency marker analysis and detailed scientific reports for our clients. If you have a scientific need for iPSC characterization, please feel free to contact us.
Reference
- Martí, M.; et al. Characterization of pluripotent stem cells. Nature protocols. 2013, 8(2): 223-253.