The previous section mostly dealt with making pluripotent stem cell lines by capturing pluripotency, harvesting pluripotent cells from the early embryo and finding ways of getting them to renew in culture without differentiating. Here we describe two alternative ways of making pluripotent stem cell lines that involve creating pluripotency by epigenetic reprogramming of the genomes of differentiated cells. That is, the pattern of DNA methylation and histone modifications in the genome of a differentiated cell is reset by artificial intervention; as a result, the chromatin structure is reset so that it resembles that found in pluripotent cells of the early embryo.
Somatic cell nuclear transfer (SCNT)
Although egg and sperm are highly-differentiated cells, they fuse to form a totipotent zygote. The cytoplasm of the egg contains factors that somehow naturally reprogram the DNA in the zygote: epigenetic marks such as repressive methylation signals are removed over large regions of the genome. Somatic cell nuclear transfer means artificially removing the nucleus of a differentiated somatic cell and placing it in an enucleated egg cell. Like the original nucleus of the egg cell, the introduced nucleus can be reprogrammed by factors in the egg cytoplasm. The egg cell with a somatic cell nucleus behaves like a zygote and can give rise to an adult organism.
When John Gurdon transferred nuclei of adult frog cells into enucleated eggs in the 1960s, he obtained a series of cloned adult frogs that were genetically identical to the original adult frog donor. But for decades afterward it was not possible to repeat this success with mammals. That is, until 1996, when a sheep called Dolly was born and became a world celebrity. The problem was that SCNT was highly inefficient in mammals. Refinements to the method were gradually introduced and a variety of other types of animal cloning followed. As detailed in Chapter 20, they have been used to introduce genetic modifications into animals to make disease models, such as a sheep model of cystic fibrosis, but they have also permitted the construction of human pluripotent stem cell lines (Figure 1).
Fig1. Constructing personalized pluripotent stem cell lines from a cloned blastocyst obtained by somatic cell nuclear transfer (SCNT). In SCNT, microsurgical techniques are used to remove a nucleus from an unfertilized egg and replace it with a nucleus taken from a differentiated somatic cell, such as a fibroblast. The introduced somatic cell nucleus has comparatively condensed chromatin but is epigenetically reprogrammed by cytoplasmic factors in the egg (red arrows) so that its pattern of chromatin conformation resembles that of a zygote. Continued development in culture allows production of a blastocyst that is genetically identical to the donor of the somatic cell. Inner cell mass cells from the blastocyst can be used to produce a pluripotent stem cell line.
Induced pluripotent stem cells (iPSC)
During development, transcription factors play key roles in determining the transition between different states of cell potency. As progenitor cells give rise to more differentiated cell lineages, the cells can simultaneously express transcription factors promoting differentiation to different cell lineages; they initially oppose each other, until one side triumphs in the tug of war. For example, cells of the undifferentiated ICM simultaneously express NANOG (promoting differentiation to epiblast) and the opposing GATA6 (promoting differentiation to the hypoblast, or primitive endoderm). Cells in which NANOG triumphs go on to become epiblast cells; those in which GATA6 triumphs become hypo blast cells.
If transcription factors drive pathways toward increasing differentiation, a question arises: Can they drive pathways in the opposite direction, toward reduced differentiation (dedifferentiation)? That question became increasingly relevant to mammalian cells after the birth of Dolly the cloned sheep: at last there was proof that mammalian cells could be epigenetically reprogrammed toward dedifferentiation, all the way to totipotency. But SCNT is technically challenging and time consuming. Could somatic differentiated cells be reprogrammed to pluripotency simply by exposing cultured cells to appropriate transcription factors? The answer was yes, and perhaps surprisingly, only four transcription factors were found to be needed (Figure 2). They included OCT4 and SOX2, two of the three master transcription factors that regulate pluripotency in vivo in the ICM, but not the third such factor, NANOG (expression of OCT4 and SOX2 is sufficient because they work together to up-regulate NANOG; see Figure 2A). KLF4 up-regulates OCT4 expression, and, like MYC, it is important in self-renewal of embryonic stem cells.
Fig2. Differentiated cells can be converted to pluripotency by artificially expressing just four transcription factors. Shinya Yamanaka and colleagues reported an astounding breakthrough in 2006 (Takahashi K & Yamanaka S [2006]; PMID 16904174) when they were able to regress cultured mouse fibroblasts to a pluripotent state by transfecting genes encoding just four types of transcription factor. (A) The starting point was 24 genes known to be important in pluripotency. They were individually cloned into retroviral expression vectors, transfected into mouse fibroblasts, and the recombinants were then cultured under conditions used to support embryonic stem cell (ESC) cultures. Pluripotent ESC-like cells could be observed because of their distinctive morphology (round, large nucleus containing large nucleoli, a thin rim of cytoplasm). (B) Not all of the 24 genes were necessary for this effect: by withdrawing some and testing different combinations, 10 genes were identified as being more important, and from this smaller set just four genes were found to be required to induce pluripotency. The four transcription factors, OCT4 (historically called Oct-3/4), SOX2, KLF4, and MYC, are sometimes known as Yamanaka factors or OSKM (from their initials).
This new type of pluripotent stem cell line came to be known as induced pluripotent stem cells (iPSCs). In 2009, viable fertile mice were reported that originated exclusively from mouse iPSCs: when introduced into blastocysts, the iPSCs can colonize the embryo to produce chimeras, and contribute to the germ line as well as to all of the somatic tissues. Although the mouse iPSCs resembled ESCs, they did exhibit some differences, notably showing the primed pluripotency reminiscent of epiblast stem cells (see Table 1). Subsequently, iPSCs have been made from more than 20 mammalian species, including humans, and different ways have been used to induce pluripotency of differentiated cells, including using purified transcription factors (instead of genes that express them), miRNAs, and small synthetic hydrocarbon molecules (selected after screening for their ability to interfere with signaling pathways).
Table1. PROPERTIES OF TWO MAJOR PLURIPOTENCY STATES IN MAMMALIAN PLURIPOTENT STEM CELL LINES
Personalized human pluripotent stem cells
Human pluripotent stem cell lines made by epigenetic reprogramming methods have one major advantage over those made by culturing cells from the early embryo: they can be made from any individual who wishes to donate easily accessible cells, such as skin fibroblasts. Pluripotent stem cell lines can therefore be made from patients and directed to differentiate to provide cells that are not readily accessible (such as neurons) for modeling disease and for drug screening. There is also the possibility of correcting genetic defects in iPSCs prepared from a patient with a genetic disorder, deriving suitable pro genitor cells, and re-introducing them into the patient so that they can be induced to differentiate into the desired cells.
