Teaching an Old Dog New Tricks: Cellular Reprogramming of Somatic Cells
Pub Date: Thursday, July 24, 2008
Author: Katja Schenke-Layland, PhD and W. Robb MacLellan, MD
Rajasingh J, Lambers E, Hamada H, Bord E, Thorne T, Goukassian I, Krishnamurthy P, Rosen KM, Ahluwalia D, Zhu Y, Qin G, Losordo DW, Kishore R., Cell-free embryonic stem cell extract-mediated derivation of multipotent stem cells from NIH3T3 fibroblasts for functional and anatomical ischemic tissue repair., Circulation research, 102 (11) e107-17. View in PubMed
The adult mammalian heart exhibits limited regenerative capacity. Consequentially, any significant loss of cardiac myocytes due to ischemia, infection, or inflammation may lead to the development of progressive heart failure and, ultimately, death. Multiple animal studies have demonstrated the ability of adult stem cells to improve left ventricular function after myocardial infarction, although documenting long-lasting effects, cardiomyocyte differentiation, or even engraftment has been more difficult. In contrast, embryonic stem (ES) cells have the capacity to robustly differentiate into all three cardiovascular cell types and have, therefore, been proposed as an alternative that could provide complete regeneration. However, major obstacles exist that have hampered the successful translation of ES cell derivatives to clinical practice, including oncogenic risk, immunological intolerance to allogeneic cells that can lead to rejection of mismatched cellular grafts, and ethical concerns surrounding their use. One solution to these limitations would be to generate autologous, genetically identical, "customized," stem cells. Consequently, investigators have been searching for ways to reprogram somatic cells into pluripotent stem cells that would have the potential to serve as an unlimited source of autologous material, which could revolutionize the treatment of heart disease.
Initial cellular reprogramming attempts were based on a technology called somatic cell nuclear transfer (SCNT), which was used to clone the famous sheep named Dolly.[3,4] In SCNT, the nucleus of a somatic cell is transplanted into an enucleated oocyte, causing the differentiated genome of the somatic cell to return to its pluripotent state. Although proof of principle that this is possible has been established, application of this technique to humans has been hampered by the inefficiency of the process and the requirement for oocytes. Thus, a groundbreaking paper, published in 2006 by Takahashi and colleagues, identifying four transcription factors whose retroviral overexpression enabled the induction of a pluripotent state in murine fibroblasts, revolutionized the field.
Investigators have now successfully reprogrammed mouse [5-7] and human [8-10] primary fibroblasts. These induced pluripotent stem (iPS) cells, although very promising, have their own set of limitations. Although thought to be similar to ES cells, their differentiation potential and full therapeutic equivalency have not been fully documented in most cases, but mouse iPS cells have been shown to be able to differentiate into functional cells of the cardiovascular system, including endothelial, smooth muscle, and cardiac myocytes.
Using an alternate approach, Rajasingh and colleagues  report on the successful reprogramming of NIH3T3 fibroblasts into cells that have the potential to differentiate into multiple cell types. These investigators were able to effect long-term changes in gene expression and genomic structure by exposing permeabilized NIH3T3 cells to extracts prepared from murine ES cells. These extracts induced dedifferentiation of NIH3T3 fibroblasts, which led to changes in cell morphology, noticeable as early as day 3 posttreatment, and an upregulation of mRNA expression of stem cell-associated transcripts including pluripotency-associated POU domain class 5 transcription factor 1 (Oct4), Nanog homeobox (Nanog), stage-specific embryonic antigen 1 (SSEA1), stem cell factor (SCF), and Kit oncogene (c-kit). Reprogrammed NIH3T3 cells also displayed epigenetic changes including CpG demethylation of the Oct4 promoter, hyperacetylation of histones 3 and 4, and decreased lysine 9 (K-9) dimethylation of histone 3 consistent with a more dedifferentiated state. When exposed to defined culture conditions, the dedifferentiated NIH3T3 cells redifferentiated in vitro into multiple cell types, although the culture conditions were not sufficient to support complete in vitro differentiation into functional cardiovascular cells. The authors further demonstrate that transplantation of the dedifferentiated NIH3T3 cells into mouse models of surgically induced hind limb ischemia or acute myocardial infarction led to significantly improved postinjury physiological function. Evidence of cell engraftment was provided, suggesting in vivo differentiation; however, the data presented cannot exclude the possibility that somatic cell fusion may account for the reported observations of plasticity of the dedifferentiated NIH3T3 cells.
Nuclear reprogramming of terminally differentiated human somatic cells into a pluripotent ES cell-like state would provide a powerful method to generate customized, patient-specific pluripotent cells for regenerative medicine efforts, assuming that reprogrammed somatic cells possess a similar differentiation potential to ES cells. Although current iPS cell technology allows the generation of patient-specific pluripotent stem cell lines , reprogramming is a rather inefficient process (typically only one in a thousand recipient cells is reprogrammed , only a few reports have focused on identifying the differentiation potential of iPS cells [11,15,16], and they are not suitable for use in human clinical studies since not only are the transcription factors themselves potentially oncogenic but the use of retroviruses to deliver the factors could generate mutations and undesirable epigenetic and transcriptional patterns in cells derived from iPS cells, ultimately leading to tumors). Thus, there is clearly a need for new approaches. The data presented by Rajasingh and colleagues are potentially promising since the authors avoid the use of potentially hazardous retroviruses while still inducing changes in cell morphology and gene expression patterns consistent with a more pluripotent state. However, the cells were not directly compared to ES cells; therefore, it is not clear if the reprogramming resulted in pluripotent cells, more similar to ES cells, or cells with a more limited differentiation potential similar to adult stem cells. In many ways, these experiments are similar to the earlier SCNT experiments suggesting that there are soluble proteins that can reprogram cells. It should be noted that SCNT was associated with a number of problems, which might also confound this technique. SCNT was an extremely inefficient process, and the efficiency of the current process was not reported by Rajasingh and colleagues. Likewise, SCNT typically results in incomplete nuclear reprogramming. Animals that have been created from pluripotent cells generated by SCNT have either died prematurely or displayed a high rate of phenotypic abnormalities. Thus, long-term studies will be required to determine the viability of cells derived using this ES cell extract-based reprogramming.
Possibly the most important question raised by the presented data is whether these results can be reproduced with primary human cells. NIH3T3 cells, the cells used for these experiments, were originally isolated from mouse embryos, so they may behave quite differently from adult and human cells. Additionally, the cells have undergone spontaneous mutations allowing them to become immortalized, which presumably would result in a less differentiated state than primary adult cells. Likewise, despite encouraging results in murine cells , the feasibility of SCNT in humans remains in question. Nonetheless, a reprogramming approach using ES cell extracts to convert differentiated somatic cells to a multipotent state could have both important basic and clinical implications. Although the mechanisms underlying the reported phenomenon of ES cell extract-induced dedifferentiation or reprogramming of NIH3T3 fibroblasts were not elucidated in the present study, when determined, this information will surely provide new insights into the process of genomic reprogramming. The identification of molecules that mediate the conversion between erasure of developmental programming of terminally differentiated somatic cell nuclei and imposition of multi- or pluripotency will be a key step toward improvement of existing protocols and toward a virus-free chemical approach to somatic cell reprogramming. Clearly, cellular reprogramming is now possible; however, it will be some time before we know its true impact on the field of regenerative medicine. Given the potentially unlimited supplies of pluripotent stem cells and reduced ethical concerns, the future of this source of cells is bright.
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-- The opinions expressed in this commentary are not necessarily those of the editors or of the American Heart Association --