Research and Development, Stemgent
Wu, D., Hamilton, B., Martin, C., Gao, Y., Ye, M., Yao, S. Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set. J. Vis. Exp. (34), e1553, doi:10.3791/1553 (2009).
In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.
2. Preparation of MEF Conditioned Medium
3. iPS Colony Selection and Passaging
4. Immunocytochemical Examination of Pluripotency Markers
Part 5: Representative Results
1. Morphology Results:
Human foreskin fibroblast (BJ) cells were co-transduced with Oct4, Sox2, Nanog, and Lin28. Morphological changes were observed as early as day 4 post-transduction, and the cluster of cells became more tightly packed at day 17 (Figure 1). Colonies were manually picked at day 25 and cultured on CF-1 MEF feeder cells. To facilitate the iPS cell colony formation after reprogramming, we used Stemolecule Y27632, a ROCK inhibitor, for the initial overnight seeding during each passage. iPS cell colonies with good morphology were observed after three sequential rounds of colony picking and passaging.
2. Expression of Pluripotency Markers:
To further characterize the isolated iPS cell colonies, we looked for the presence of common pluripotency markers expressed in ES cells. The colonies exhibited strong alkaline phosphatase (AP) activity (Figure 2). Additionally, immunocytochemistry (ICC) was performed on the iPS cell colonies with a panel of pluripotency marker-specific antibodies, including surface markers TRA-1-81, TRA-1-60, SSEA-4 and SSEA-3 as well as nuclear markers Oct4, Sox2 and Nanog. The isolated iPS colonies were positive for all markers (Figure 3). The ICC results show that the iPS cells exhibited the appropriate pluripotency marker expression pattern, demonstrating that these iPS cells closely resemble undifferentiated human ES cells.
Figure 1: Morphological changes of transduced BJ cells. Bright field images of a typical iPS cell colony formed at (A) 4 days and (B) 17 days post-transduction.
Figure 2: AP activity of reprogrammed BJ cells. Three different colonies stained with Stemgent Alkaline Phosphatase Staining Kit.
Figure 3: Human iPS cells express high levels of the following ES cell specific markers: surface markers TRA-1-81, TRA-1-60, SSEA-4 and SSEA-3, and nuclear markers Oct 4, Nanog and Sox2.
These results demonstrate that the Stemgent Human TF Lentivirus Set can be used to efficiently generate iPS colonies by inducing the ectopic expression of transduced transcription factors in human fibroblasts. When designing reprogramming experiments, several variables should be considered to optimize the efficiency of reprogramming. First, the active virus-to-target ratio (multiplicity of infection M.O.I.) may need to be modified during the primary transduction step to achieve optimum transduction efficiency. Second, the growth condition of the target cells can impact reprogramming. Healthy and proliferative cells are more amenable to reprogramming. Third, when modifying the protocol for different cell numbers, it is recommended that target cell numbers be adjusted proportionally to the surface area of the culture dish. Lastly, applying ROCK inhibitors such as Y27632 should be considered to help ensure successful reprogramming as recent studies have demonstrated its utility in enhancing hES colony survival.4,5
The authors of this article are employed by Stemgent that produces reagents and instruments used in this article.
|Human TF Lentivirus Set||Stemgent||00-0005|