LIF-Dependent Porcine iPSCs | T2503

Leukemia inhibitory factor-dependent induced pluripotent stem cells were derived from the inner cell mass of porcine blastocysts by up-regulating expression of KLF4 and POU5F1. They are dependent on leukemia inhibitory factor signaling for maintenance of pluripotency, can be cultured over extended passage, and have the ability to form teratomas. Resulting colonies resemble mouse embryonic stem cells in terms of colony morphology, cell cycle interval, transcriptome, and the expression of pluripotent markers. This cell line was created by lentiviral transduction with hKLF4 and M2rtTA virus.

Vector:

N/A

Species:

Pig (Porcine)

Organ:

Embryo

Passages:

Propagation:

The base medium for this cell line is Prigrow IV medium available at abm, Cat. No: TM004. To make the complete growth medium, add the following components to the base medium: Neurbasal medium (Invitrogen) to a final concentration of 49%, N2 supplement (100X) (Invitrogen) to a final concentration of 0.5%, B27 supplement (50X) (Invitrogen), 3i inhibitor cocktail (see following), human LIF (2000 units/uL) (Genscript) to a final concentration of 0.05%, L-glutamine-BME (L-glutamine to 99.86% and 2-mercaptoethanol to 0.14%) to a final concentration of 0.5%, non-essential amino acids (100X) (Sigma) to a final concentration of 1%, protease-free BSA (5%) (Sigma) to a final concentration of 0.2%, and doxycycline hyclate (4mg/mL) (Sigma) to a final concentration of 0.05%. 3i inhibitor cocktail can be prepared as follows:370 uL DMSO, 100uL CHIR99021 (GSK3 beta inhibitor) 30mM stock (Stemgent), 20uL PD032591 (MEK inhibitor) 40 mM stock (Stemgent), 10uL PD173074(FGFR inhibitor) 10 mM stock (Sigma).
Change media every 2-3 days.
Carbon dioxide (CO₂): 5%, Temperature: 37.0°C.
* It is not recommended to use heat-inactivated FBS for cell culture unless specified otherwise.

Concentration:

10⁶ cells/vial

Induction Method:

N/A

Pluripotency:

N/A

Growth Properties:

Adherent

Morphology:

Polygonal

Markers:

Pluripotent markers, such as POU5F1, SOX2, and surface marker SSEA1.

Subculturing:

1:10 split, Accutase (Millipore) used

QC:

1) Reverse transcriptase PCR; 2) Real time PCR; 3) Transcriptional profiling by microarray; 4) Alkaline phosphatase staining; 5) Immunofluorescence; 6) Western blot; 7) Karyotyping

Safety:

BioSafety Level II

Shipping:

Dry Ice

Storage:

-180°C