Services

Characterisation of pluripotency status, functional pluripotency and cell identity

The pluripotency status as well as functional pluripotency are confirmed by FACS and immunofluorescent staining, respectively. Matching identity of primary cells and hiPSCs is confirmed by STR analysis. The number of characterised clones is determined by the customer.

Costs per clone:

                                                                       LUMC                                       External academia

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Costs per clone:

                                                                       LUMC                                       External academia

Pluripotency status, morphology,

functional pluripotency, STR                      €82                                           €192

Karyotyping

ddPCR assay StemGenomics*                    €209                                         €345

G-banding**                                                €253                                         €376

 

* ICS digital PSC 24 probes assay; www. stemgenomics.com

** 20 metaphases

Characterisation of pluripotency status and functional pluripotency of hiPSCs

Characterisation of hiPSCs for quality control (QC)  

hiPSC lines are characterised as follows:

Morphology

Brightfield images of representative hiPSC colonies.

Pluripotency status

The percentage of undifferentiated cells expressing SSEA4, Nanog and OCT3/4 will be determined by FACS analysis. hiPSCs pass QC when ≥ 70% express SSEA4/Nanog and SSEA4/OCT3/4.

Functional pluripotency

hiPSCs are differentiated short-term using the STEMdiff Trilineage Differentiation Kit (STEMCELL Technologies). The presence of derivatives of ecto-, endo- and mesoderm is analysed by immunofluorescent (IF) staining with 3 markers per germ layer: Pax6, Nestin, FABP7 (ectoderm); CDX2, Vimentin, Brachyury T (mesoderm); FOXA2, GATA4, EOMES (endoderm). hiPSCs pass QC when at least 2 markers per germ layer are present.

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hiPSC lines are characterised as follows:

Morphology

Brightfield images of representative hiPSC colonies.

Pluripotency status

The percentage of undifferentiated cells expressing SSEA4, Nanog and OCT3/4 will be determined by FACS analysis. hiPSCs pass QC when ≥ 70% express SSEA4/Nanog and SSEA4/OCT3/4.

Functional pluripotency

hiPSCs are differentiated short-term using the STEMdiff Trilineage Differentiation Kit (STEMCELL Technologies). The presence of derivatives of ecto-, endo- and mesoderm is analysed by immunofluorescent (IF) staining with 3 markers per germ layer: Pax6, Nestin, FABP7 (ectoderm); CDX2, Vimentin, Brachyury T (mesoderm); FOXA2, GATA4, EOMES (endoderm). hiPSCs pass QC when at least 2 markers per germ layer are present.

Cell line authentication

One hiPSC line per donor sample and the corresponding primary cells are subjected to short tandem repeat (STR) analysis to confirm matching identity. This service is performed by the LUMC department of Human Genetics.

Upon completion of characterisation the customer is provided with:

  • Cryovials for each characterised hiPSC line at 2 passages (3 vials each)
  • Cryovials for each uncharacterised hiPSC line at 1 passage (2 vials)
  • Summary of characterisation/ QC data (PowerPoint; raw data upon request)

Standard Operation Procedure (SOP) for starting up cells