C-PEP-mCherry-hiPSC

General#

Cell Line

hPSCreg Name HMGUi001-A-8
Alternative name(s)
C-PEP-mCherry-hiPSC
Cell line type Human induced pluripotent stem cell (hiPSC)
Last update 22nd September 2020
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Provider

Generator Institute of Diabetes and Regeneration Research (IDR)
Owner Institute of Diabetes and Regeneration Research (IDR)
Distributors
Derivation country Germany

External Databases

BioSamples SAMEA7249858

General Information

* Is the cell line readily obtainable for third parties?
No
Subclone of

Donor Information#

General Donor Information

Sex female
Ethnicity Caucasian

Phenotype and Disease related information (Donor)

Diseases No disease was diagnosed.
Disease associated phenotypes no phenotypes
Family history no
Is the medical history available upon request? no
Is clinical information available? no

Karyotyping (Donor)

Has the donor karyotype been analysed?
No

Other Genotyping (Donor)

Is there genome-wide genotyping or functional data available?
No

External Databases (Donor)

BioSamples SAMEA5696688

Ethics#

Also have a look at the ethics information for the parental line HMGUi001-A .
Is there an MTA available for the cell line? No
For generation of the cell line, who was the supplier of any recombined DNA vectors or commercial kits used? Vectors were generated at IDR, pU6-(BbsI)-sgRNA-CAG-Cas9-Venus-bpA plasmid (Addgene plasmid #86986) was used.
Are you aware of any constraints on the use or distribution of the cell line from the owner or any parties identified in the query above? Yes
Constraints for use or distribution According to donor consent the line must not be used for commercial purposes. For research purposes project-specific review by a medical Ethics Committee is obligatory.

hIPSC Derivation#

General

The source cell information can be found in the parental cell line HMGUi001-A.
Passage number reprogrammed 23

Reprogramming method

Vector type Non-integrating
Vector HMGUi001-A was targeted with two vectors. For the first vector, the Cas9-Venus-sgRNA vector, a sgRNA was cloned into the pU6-(BbsI)-sgRNA-CAG-Cas9-Venus-bpA plasmid (Addgene plasmid #86986). Cas9 and a targeting site specific sgRNA were expressed from this vector to induce a double strand break at the targeting region. The second vector contained the template for the intended homology directed repair: the mCherry coding sequence flanked by a 1111 bp left homology arm (5’ HA) and a 901 bp right homology arm (3’ HA). Silent mutations in the sgRNA binding site were introduced to avoid cutting of the correctly inserted sequences.

Vector free reprogramming

Other

Selection criteria for clones Transfected cells expressed Venus and were sorted by FACS. The genotype of the clones was screened by PCR and verified by Sanger sequencing.
Derived under xeno-free conditions
Yes
Derived under GMP?
No
Available as clinical grade?
No

Culture Conditions#

Surface coating Matrigel/Geltrex
Feeder cells
No
Passage method EDTA
O2 Concentration 21 %
CO2 Concentration 5 %
Medium Other medium:
Base medium: StemMACS iPS-Brew XF medium
Main protein source: xeno-free
Serum concentration: 0 %
Has Rock inhibitor (Y27632) been used at passage previously with this cell line?
Yes
Has Rock inhibitor (Y27632) been used at cryo previously with this cell line?
Yes
Has Rock inhibitor (Y27632) been used at thaw previously with this cell line?
Yes

Characterisation#

Analysis of Undifferentiated Cells
Marker Expressed Immunostaining RT-PCR FACS Enzymatic Assay Expression Profiles
SOX2
Yes
POU5F1 (OCT-4)
Yes
SSEA-4
Yes
TRA 1-60
Yes
Oct-3
Yes
Differentiation Potency
Endoderm
Ont Id: UBERON_0000925
Marker Expressed
SOX17
Yes
FOXA2
Yes
Mesoderm
Ont Id: UBERON_0000926
Marker Expressed
SM22-alpha
Yes
Ectoderm
Ont Id: UBERON_0000924
Marker Expressed
Nestin
Yes

Microbiology / Virus Screening

Mycoplasma Negative

Genotyping#

Karyotyping (Cell Line)

Has the cell line karyotype been analysed?
Yes
46, XX
Karyotyping method: G-Banding

Other Genotyping (Cell Line)

Genetic Modification#

Genetic modifications not related to a disease
insulin (target)
Variant
Chromosome 11, insulin gene, exon 3
Heterozygous
The C-PEP-mCherry-hiPSC line presented here was generated by heterozygous insertion of the mCherry sequence at the C-terminus of the C-peptide, which is encoded by exon 3 of the insulin locus.