1. ¹The Institute for Cell Engineering, Department of Gynecology & Obstetrics, the Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
2. ²Reproductive Medicine Center, The 1st Affiliated Hospital of Zhongshan University, Guangzhou 510089, China
3. ³Graduate Immunology program, the Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
4. ⁴Graduate Program of Biomedical Engineering, the Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

Correspondence: Linzhao Cheng, Stem Cell Program, Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Broadway Research Building, Room 747, 733 North Broadway, Baltimore, MD 21205, USA Tel: 1-410-614-6958; Fax: 1-443-287-5611 E-mail: lcheng@welch.jhu.edu

^*These two authors contributed equally to this work.

⁵Current address: Whitehead Institute for Biomedical Research, Cambridge, MA, 02142, USA

Received 26 September 2006; Revised 18 December 2006; Accepted 22 December 2006; Published online 9 January 2007.

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.