human recombinant leukemia inhibitory factor hrlif  (R&D Systems)

Journal: Cells

Article Title: Controlling the Switch from Neurogenesis to Pluripotency during Marmoset Monkey Somatic Cell Reprogramming with Self-Replicating mRNAs and Small Molecules

doi: 10.3390/cells9112422

Figure Lengend Snippet: Generation of marmoset induced pluripotent stem cells (iPSCs) with the Tomato-modified self-replicating mRNA based on the Venezuelan equine encephalitis virus (VEE-OKS-iM-iTomato). ( A ) Structure of the reprogramming VEE-OKS-iM-iTomato mRNA. ( B ) Scheme of the reprogramming process. ( C ) Image of marmoset fetal fibroblasts (cjFFs) transfected with VEE-OKS-iM-iTomato at day 3 post-transfection. Transfected cells are recognizable by their red fluorescence. ( D ) An intermediate primary colony at day 27 post-transfection with red VEE-OKS-iM-iTomato fluorescence. ( E ) An intermediate primary colony at day 27 post-transfection with characteristic compact morphology and clearly defined borders (indicated with arrowheads). The cells beyond the upper and left borders of the colony are non-reprogrammed cjFFs. ( F ) An intermediate primary colony stained for alkaline phosphatase (AP). ( G ) Marmoset iPSC colony after second round of reprogramming of the intermediate primary cells with IWR1, CHIR99021, CGP77675, human recombinant leukemia inhibitory factor (hrLIF), and Forskolin. ( H ) Morphology of marmoset iPSC colonies growing on Geltrex at P27. (All scale bars = 200 μm).

Article Snippet: The cell suspension was seeded in 6 cm Geltrex dishes (30–40 × 10 5 cells/dish) in marmoset iPSC medium (iPS-Brew supplemented with 3 μM IWR1 (Sigma-Aldrich, Munich, Germany), 0.5 μM CHIR99021, 0.7 μM CGP77675 (Selleckchem, Houston, TX, USA), 10 ng/mL human recombinant leukemia inhibitory factor (hrLIF) (PeproTech, Hamburg, Germany), and 7 μM Forskolin (Selleckchem, Houston, TX, USA)) and cultured for 2–3 passages in hypoxic conditions (5% O 2 , 5% CO 2 , and 90% N 2 ) until the appearance of colonies with iPSC-like morphology.

Techniques: Modification, Virus, Transfection, Fluorescence, Staining, Recombinant

Journal: Cells

Article Title: Controlling the Switch from Neurogenesis to Pluripotency during Marmoset Monkey Somatic Cell Reprogramming with Self-Replicating mRNAs and Small Molecules

doi: 10.3390/cells9112422

Figure Lengend Snippet: Role of small molecule inhibitors in maintaining pluripotency gene expression of marmoset iPSCs in long-term culture. ( A ) Marmoset iPSCs cultured either with full culture medium, with omission of individual factors (-IWR1, -CHIR99021, -CGP77675, -hrLIF, -Forskolin), or without any inhibitors and hrLIF. (All scale bars = 50 μm). ( B ) Pluripotency gene expression of marmoset iPSCs cultured without different inhibitors or hrLIF for 5–6 passages determined by relative quantitation qPCR. One of the lines cultured without any inhibitors and hrLIF was used as a reference. (Data are presented as mean + SEM. Statistically significant differences between experimental groups are indicated with asterisks as follows: * p < 0.05, ** p < 0.01).

Article Snippet: The cell suspension was seeded in 6 cm Geltrex dishes (30–40 × 10 5 cells/dish) in marmoset iPSC medium (iPS-Brew supplemented with 3 μM IWR1 (Sigma-Aldrich, Munich, Germany), 0.5 μM CHIR99021, 0.7 μM CGP77675 (Selleckchem, Houston, TX, USA), 10 ng/mL human recombinant leukemia inhibitory factor (hrLIF) (PeproTech, Hamburg, Germany), and 7 μM Forskolin (Selleckchem, Houston, TX, USA)) and cultured for 2–3 passages in hypoxic conditions (5% O 2 , 5% CO 2 , and 90% N 2 ) until the appearance of colonies with iPSC-like morphology.

Techniques: Gene Expression, Cell Culture, Quantitation Assay