EDi001-A-4

Induced pluripotent stem cells (iPSCs) are adult cells that have been reprogrammed to an embryonic stem cell–like state. The cells can replicate indefinitely or, under controlled conditions, can be differentiated into any other cell type such as nerve, heart or liver cells. Medical researchers are able to use iPS cells to test how different patients might respond to new drugs or to analyse how genetic diseases develop.

The EBiSC stem cell bank is a collection of human iPS cells available to academic and commercial researchers for use in disease modelling and other forms of stem cell research. The initial collection has been generated from a wide range of donors representing specific disease backgrounds and healthy controls. EBiSC has established many routine procedures for collecting, expanding and characterizing human iPS cell lines. The stem cell bank includes iPSC cell lines derived from neurodegenerative diseases (Alzheimer′s Disease, Parkinson′s Disease, Dementia, Motor Neuron Disease (ALS) - and Huntington′s Disease), eye and heart diseases, and lines from healthy control donors for age and sex matching.

Depositor
University of Edinburgh

Derivation
Primary cell type: -
Reprogramming method
Vector type: Integrating
Vector: Virus
Virus type: Retrovirus
Gene list:
KLF4
MYC
POU5F1
SOX2
Have the reprogramming vectors been silenced: Yes
Merthods used: rtpcr

Xeno free conditions: no
Derived under gmp: no
Available as clinical grade: no

Characterization
Analysis of Undifferentiated Cells
Marker expression:
Marker Expressed Immunostaining RT-PCR FACS Enzymatic Assay Expression Profiles
TRA 1-60 Yes
Microbiology / Virus Screening
HIV 1: Not done
HIV 2: Not done
Hepatitis B: Not done
Hepatitis C: Not done
Mycoplasma: Negative
Sterility
Inoculation for microbiological growth: No Contaminants Detected
Mycoplasma: Not Detected
Viability: Viable post-cryopreservation

Genotyping
STR/Fingerprinting: A 16 allele profile has been recorded and data is available upon request, after cell line purchase.

Genetic Modification
Disease/phenotype related modifications
Disease: Parkinson′s disease
Type of modification: Gene knock-out
Gene: SNCA
Chromosome location: 4q22.1
Delivery method: CRISPR-associated (CRISPR/Cas) System
Description: The SCNA triplication has been gene edited to become *putatively* normal. CRISPR/Cas was employed to create a mutation in Exon2, disrupting the ATG start site and some sequence 5′ to the coding start of SCNA. PCR amplicons of the CRISPR site were cloned using the TOPO cloning technique. Sequencing of 8 TOPO clones detected 2 deletions, both of which disrupt the first ATG start site. This indicates 2 alleles were putatively knocked out, and 2 alleles remain. Additional comment: there are two additional ATG sites located immediately downstream of the normal ATG start site, one of which is in-frame (9 bases) to the initial ATG. It is possible that inactivation of the first ATG start site might not be completely sufficient to prevent translation from the second downstream ATG, which is in-frame to the first.

Medium: mTeSR^®
Passage method: EDTA
Matrix: Matrigel^® / Geltrex^®
CO₂ concentration: 5%
O₂ concentration: 20%
Temperature: 37°C

Note: EAUA and CLIP must be completed before order fulfillment

EBiSC is a trademark of Fraunhofer-Gesellschaft

GELTREX is a registered trademark of Life Technologies Corporation

Matrigel is a registered trademark of Corning, Inc.

mTeSR is a registered trademark of WiCell Research Institute, Inc.