Increases in reprogramming efficiency owed to increased understanding of underlying biology

by Lisa Girard, PhD
HSCI Science Editor

The ability to generate embryonic stem cell-like cells from somatic cells by reprogramming holds tremendous potential for reshaping our approaches to regenerative medicine, drug discovery, and understanding disease mechanisms. Nuclear reprogramming is a powerful means by which to create patient-specific cell lines. These lines can provide an immunologically-compatible source of cells for regenerative approaches, as well as enable the creation of disease specific cell lines from patients that can provide enough material for large scale screens and studies. This is particularly advantageous for instances in which affected cell types are difficult to obtain in quantities sufficient for study, such as neurodegenerative disorders. The approaches shown to be able to reprogram somatic cells include cell fusion, somatic cell nuclear transfer, and the creation of induced pluripotent stem (iPS) cells through the introduction of defined factors (for a review see Yamanaka, 2007). iPS cell reprogramming has received a great deal of attention within the past year, with the publication of iPS cells generated from human somatic cells (Takahashi et al., 2007; Yu et al., 2007; Park et al., 2008) and subsequent proof of principle experiments (Hanna et al., 2007) using iPS cells generated from the tail skin cells of a sickle cell anemia mouse model to produce healthy blood cells which they then showed could cure the mouse. iPS cell-based approaches have also garnered much media attention since they circumvent many of the ethical arguments currently put forth by opponents of embryonic stem cell research (see Hyun et al., 2007 for related discussion).

While reprogramming has the potential to be of great therapeutic value, many existing issues currently create a chasm between the laboratory bench and the clinic. The reprogramming process is inefficient, currently with a transformation efficiency of only approximately .1% (Maherali et al., 2007; Wernig et al., 2007; Takahashi and Yamanaka, 2006). Also, the viral vectors used to introduce the iPS cell reprogramming factors are potentially oncogenic, as are some of the factors themselves. Reprogramming offers an opportunity to better understand the process of differentiation and development, providing an in vitro recapitulation of events. This better understanding, in turn, informs our efforts to optimize such protocols for therapeutic use. Illustrating this, a better understanding of the factors and chronology of events would allow us to increase the efficiency of iPS cell recovery. Marker selection to isolate iPS cells initially used expression of the ES cell-specific factor Fbx15. It was then determined not long after that the factors Oct4 and Nanog are actually much more effective markers for isolating cells with properties apparently indistinguishable from embryonic stem cells (Maherali et al., 2007; Wernig et al., 2007; Okita et al., 2007), revealing molecular signatures for the progression of events. Understanding more completely this temporal landscape of reprogramming could refine the selection process further, increasing the efficiency of iPS cell recovery.

The recent identification of intermediate cell populations in the reprogramming process (Bambrink et al., 2008; Stadfeld et al., 2008) is beginning to open the black box that largely exists between the introduction of iPS cell factors and the emergence of pluripotent cells in culture. The key parameters being defined include more precisely when, and for what duration particular factors are needed. Two groups independently conducted their investigations using an antibiotic-inducible control system for iPS cell factor expression. The degree of control this system provided allowed them to identify which factors, when downregulated or upregulated, are indicative of pluripotency. Furthermore, the inducible system revealed the window of necessity and sufficiency for these factors. The significance of these findings is, at a minimum, two-fold. Not only does it begin to let us understand the biology of the seemingly nebulous reprogramming process, but it also, very significantly, moves us closer to transitioning from viral vector-based constitutive expression of iPS cell factors to more tightly controlled expression systems which are more tractable for therapeutic applications. Additionally, the information lets us identify the most optimal selectable markers for successful iPS cell transformation based on our growing knowledge of which factors are expressed toward the end of the progression toward pluripotency, thus allowing selection based upon criteria of the highest stringency.

The advent of nuclear reprogramming with iPS cell factors arrived with a splash. It is only now that we are taking a step back and examining how the observed phenomena informs our understanding of the biology that we can appreciate the true impact of the findings and ultimately move forward.

References

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