Recently, it was found that overexpression of KLF4, in combination with three other transcription factors could transform mouse fibroblasts into a state resembling embryonic stem cells (ES cells). These cells have been termed “inducible pluripotent stem cells” (iPS cells) [78
]. By replacing the open reading frame of Fbx15
, a non-essential marker of embryonic stem cells, with a neomycin resistance gene, it was hypothesized that neomycin-resistant colonies might have somehow reprogrammed themselves into embryonic stem cells. After screening a short list of potential factors, it was found that the simultaneous infection of retroviruses expressing Oct3/4, Sox2, c-Myc, and KLF4 were able to produce resistant clones. These cells could form teratomas that contained differentiated tissues from all three germ layers, confirming their pluripotency. This approach was further refined by screening for neomycin resistance based on Nanog
expression instead of Fbx15.
Unlike Fbx15-iPS cells, Nanog and Oct4-iPS could produce chimeric mice and, could generate live late-term embryos when injected into tetraploid blastocysts [79
]. Thus, Nanog and Oct4-iPS satisfy are even more stringent tests of pluripotency than Fbx15-iPS cells.
An area currently under intense investigation is understanding the molecular events that occur during stem cell reprogramming as well as the precise role of each of the four individual factors required. The importance of Oct3/4 and Sox2 in ES cell renewal is well established [82
]. What is less clear is the function of the other two factors that make up the “magic brew”: c-Myc and KLF4. One possibility is that c-Myc and KLF4 confer increased proliferative capacity on potential iPS cells, since both can function as oncogenes [83
]. Since c-Myc regulates a significant number of genes, its function may be to effect global changes in the chromatin environment by recruiting histone acetyl-transferase complexes. According this model, KLF4 may then function to inhibit apoptosis induced by overexpression of c-Myc. KLF4 represses expression of c-Myc expression in colon cancer cells through inhibiting Wnt signaling [10
]. Thus, c-Myc may provide a balance for KLF4. The role of Wnt signaling in iPS cells is still an open question.
Overexpression of KLF4 in ES cells inhibited differentiation into erythroid progenitors, and increased their capacity to generate secondary embryoid bodies, suggesting a role for KLF4 in self-renewal [84
]. In concert with Oct3/4 and Sox2, KLF4 activates expression of Lefty1
, a gene expressed in ES cells, but lost during differentiation [85
]. In addition, KLF4-null mice survive to term and have no detectable defects during embryogenesis in their pluripotent stem cell population [11
], suggesting that in normal ES cells, KLF4 may be dispensable. More recently, human iPS have been produced using a slightly different mix of factors, substituting c-Myc and KLF4 with Nanog and LIN28 [86
], further calling into question the overall importance of c-Myc and KLF4. It has even been suggested that c-Myc and KLF4 are merely molecular catalysts, in that they might accelerate or increase the efficiency of the reprogramming process, but are otherwise not absolutely required [87
However, a recent study has found that the function in ES cell self-renewal of KLF4 is partially redundant with KLF2 and KLF5, as knockdown of all three Krüppel-like factors, but not any one individually, resulted in spontaneous ES cell differentiation [88
]. In addition, significant overlap was found between genes regulated by Nanog and the three Krüppel-like factors. Clearly, a complete understanding of the role of KLF4 in ES cell self-renewal and iPS cell reprogramming awaits further study