Promoting human embryonic stem cell renewal or differentiation by modulating Wnt signal and culture conditions

Authors:
Cai L, Ye Z, Zhou BY, Mali P, Zhou C, Cheng L
In:
Source: Cell Res
Publication Date: (2007)
Issue: 17(1): 62-72
Research Area:
Cancer Research/Cell Biology
Cells used in publication:
Embryonic Stem Cell (ES), human
Species: human
Tissue Origin: embryo
Platform:
Nucleofector™ I/II/2b
Experiment
Both enhanced green fluorescent protein (GFP) and and improved red fluorescent protein (RFP) called tandem dimer (td) Tomato, also cDNA3.1-based plasmid expressing tdTomato RFP reporter, containing a neomycin resistance gene controlled by the SV40 promoter were used. The circular plasmid (5 µg) was transfected into about 2 mllion hES cells using Amaxa Mouse ES solution and program A-23 (Nucleofector II). Treated cells were plated into 3 wells of HAFI/Wnt3a feeder cells (in a 6-well plate, after 40 GY irradiation). Two days after, 100 µg/ml G418 were added. Transfected cells were passaged onto fresh HAFI/Wnt3a cells as needed.
Abstract
We previously showed that Wnt3a could stimulate human embryonic stem (hES) cell proliferation and affect cell fate determination. In the absence of feeder cell-derived factors, hES cells cultured under a feeder-free condition survived and proliferated poorly. Adding recombinant Wnt3a in the absence of feeder cell derived-factors stimulated hES cell proliferation but also differentiation. In the present study, we further extended our analysis to other Wnt ligands such as Wnt1 and Wnt5a. While Wnt1 displayed a similar effect on hES cells as Wnt3a, Wnt5a had little effect in this system. Wnt3a and Wnt1 enhanced proliferation of undifferentiated hES cells when feeder-derived self-renewal factors and bFGF are also present. To explore the possibility to promote the proliferation of undifferentiated hES cells by activating the Wnt signaling, we overexpressed Wnt3a or Wnt1 gene in immortalized human adult fibroblast (HAFi) cells that are superior in supporting long-term growth of undifferentiated hES cells than primary mouse embryonic fibroblasts. HAFi cells with or without a Wnt transgene can be propagated indefinitely. Over-expression of the Wnt3a gene significantly enhanced the ability of HAFi feeder cells to support the undifferentiated growth of 3 different hES cell lines we tested. Co-expression of three commonly-used drug selection genes in Wnt3a-overpressing HAFi cells further enabled us to select rare hES clones after stable transfection or transduction. These immortalized engineered feeder cells (W3R) that co-express growth-promoting genes such as Wnt3a and three drug selection genes should empower us to efficiently make genetic modified hES cell lines for basic and translational research.