Generation of induced pluripotent stem cells by reprogramming human fibroblasts with the stemgent human TF lentivirus set.
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.
Wu
Dongmei
D
Research and Development, Stemgent.
Hamilton
Brad
B
Martin
Charles
C
Gao
Yan
Y
Ye
Mike
M
Yao
Shuyuan
S
D000818:Animals; D004318:Doxycycline / Q000494:pharmacology; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D000090062:Kruppel-Like Factor 4; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D039904:Pluripotent Stem Cells / Q000166:cytology*; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics; D014161:Transduction, Genetic / Q000379:methods*
C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D014157:Transcription Factors; D004318:Doxycycline