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Keywords:

  • nuclear transplantation;
  • pluripotent;
  • stem cells;
  • transcription factors

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Nuclear transplantation
  5. Fusion of somatic cells and embryonic stem cells
  6. Reprogramming by defined transcription factors
  7. A ‘reprogrammable’ mouse: the ‘secondary’ system
  8. Reprogramming of somatic cells by a single polycistronic vector
  9. Therapeutic potential of iPS cells
  10. Outlook
  11. Acknowledgments
  12. Disclosure of Conflict of Interests
  13. References

Summary.  Reprogramming of somatic cells to a pluripotent embryonic stem cell-like state has been achieved by nuclear transplantation of a somatic nucleus into an enucleated egg and most recently by introducing defined transcription factors into somatic cells. Nuclear reprogramming is of great medical interest as it has the potential to generate a source of patient-specific cells. This short review summarizes strategies to reprogram somatic cells to a pluripotent embryonic state and discuss the implications of this technology for transplantation medicine.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Nuclear transplantation
  5. Fusion of somatic cells and embryonic stem cells
  6. Reprogramming by defined transcription factors
  7. A ‘reprogrammable’ mouse: the ‘secondary’ system
  8. Reprogramming of somatic cells by a single polycistronic vector
  9. Therapeutic potential of iPS cells
  10. Outlook
  11. Acknowledgments
  12. Disclosure of Conflict of Interests
  13. References

All mammalian somatic cells originate from a single fertilized cell, the zygote, and share identical genetic information despite the dramatic changes in cell structure and function that accompany organismal development. The genome is subjected to a wide array of epigenetic modifications during lineage specification, a process that contributes to the implementation and maintenance of specific gene expression programs in somatic cells [1]. Nuclear transfer and cell-fusion experiments demonstrate that the epigenetic signature directing a cell identity can be erased and modified into that of another cell type. Furthermore, in the case of cloning, differentiated cells can be reprogrammed back to pluripotency to support the re-expression of all developmental programs. Indeed, the generation of cloned animals from somatic cell nuclei proved beyond doubt that major genetic changes that would prevent a somatic nucleus from generating all tissue types are not part of the normal developmental process. A major advance in the stem-cell field has been the success to in vitro convert somatic cells back to an embryonic state by the use of defined transcription factors [2]. In the following pages, I will summarize the main strategies to achieve reprogramming.

Nuclear transplantation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Nuclear transplantation
  5. Fusion of somatic cells and embryonic stem cells
  6. Reprogramming by defined transcription factors
  7. A ‘reprogrammable’ mouse: the ‘secondary’ system
  8. Reprogramming of somatic cells by a single polycistronic vector
  9. Therapeutic potential of iPS cells
  10. Outlook
  11. Acknowledgments
  12. Disclosure of Conflict of Interests
  13. References

Nuclear cloning provided proof for the notion that irreversible alterations of the genome are not required for normal development. However, because no genetic marker was available in the initial cloning experiments, it remained an open question whether terminally differentiated cells could be reprogrammed to a totipotent state. The successful generation of cloned mice from genetically marked lymphoid cells [3] or from post mitotic neurons [4,5] unambiguously demonstrated that terminal differentiation does not restrict the potential of the nucleus to support development. Cloning from terminally differentiated donor cells is, however, inefficient and was in many instances successful only when a ‘two-step’ procedure, which involved the generation of cloned ES cells as an intermediate, was used.

Fusion of somatic cells and embryonic stem cells

  1. Top of page
  2. Abstract
  3. Introduction
  4. Nuclear transplantation
  5. Fusion of somatic cells and embryonic stem cells
  6. Reprogramming by defined transcription factors
  7. A ‘reprogrammable’ mouse: the ‘secondary’ system
  8. Reprogramming of somatic cells by a single polycistronic vector
  9. Therapeutic potential of iPS cells
  10. Outlook
  11. Acknowledgments
  12. Disclosure of Conflict of Interests
  13. References

Epigenetic reprogramming of somatic nuclei to an undifferentiated state has been demonstrated in murine hybrids produced by fusion of embryonic cells with somatic cells. Hybrids between various somatic cells and embryonic carcinoma cells [6], embryonic germ (EG) or ES cells [7] share many features with the parental embryonic cells indicating that the pluripotent phenotype is dominant in such fusion products. As with mice [8], human ES cells have the potential to reprogram somatic nuclei after fusion [9,10].

Reprogramming by defined transcription factors

  1. Top of page
  2. Abstract
  3. Introduction
  4. Nuclear transplantation
  5. Fusion of somatic cells and embryonic stem cells
  6. Reprogramming by defined transcription factors
  7. A ‘reprogrammable’ mouse: the ‘secondary’ system
  8. Reprogramming of somatic cells by a single polycistronic vector
  9. Therapeutic potential of iPS cells
  10. Outlook
  11. Acknowledgments
  12. Disclosure of Conflict of Interests
  13. References

Takahashi and Yamanaka recently achieved a significant breakthrough in reprogramming somatic cells back to an ES-like state [11]. They successfully reprogrammed mouse embryonic fibroblasts (MEFs) and adult fibroblasts to pluripotent ES-like cells after viral-mediated transduction of the four transcription factors Oct4, Sox2, c-myc and Klf4 followed by selection for activation of the Oct4 target gene Fbx15. Cells that had activated Fbx15 were designated with a coined expression, i.e., iPS (induced pluripotent stem) cells. These cells were shown to be pluripotent by their ability to form teratomas although they were unable to generate live chimeras. In subsequent experiments when activation of the endogenous Oct4 or Nanog genes was used as a more stringent selection criterion for pluripotency, the resulting Oct4-iPS or Nanog-iPS cells, in contrast to Fbx15-iPS cells, were fully reprogrammed to a pluripotent, ES cell state by molecular and biological criteria [12–14]. Shortly after the reprogramming of mouse cells had been achieved the generation of iPS cells from human fibroblasts was reported [15,16].

While genetic experiments have established that Oct4 and Sox2 are essential for pluripotency [17], the role of the two oncogenes, c-myc and Klf4, in reprogramming is less clear. Some of these oncogenes may, in fact, be dispensable for reprogramming as both mouse and human iPS cells have been obtained in the absence of c-myc transduction, although with low efficiency [16,18,19].

A ‘reprogrammable’ mouse: the ‘secondary’ system

  1. Top of page
  2. Abstract
  3. Introduction
  4. Nuclear transplantation
  5. Fusion of somatic cells and embryonic stem cells
  6. Reprogramming by defined transcription factors
  7. A ‘reprogrammable’ mouse: the ‘secondary’ system
  8. Reprogramming of somatic cells by a single polycistronic vector
  9. Therapeutic potential of iPS cells
  10. Outlook
  11. Acknowledgments
  12. Disclosure of Conflict of Interests
  13. References

Reprogramming by defined transcription factors is an inefficient process impeding mechanistic studies of this process. To overcome these obstacles, we devised a drug-inducible transgenic system, termed ‘secondary system’, which supports reprogramming of multiple somatic cell types carrying identical Dox-inducible viral transgenes encoding Oct4, Sox2, Klf4, and c-Myc [20,21]. This was achieved by infecting fibroblasts with Dox-inducible lentiviral vectors carrying the four reprogramming factors [22,23]. When cultured in the presence of Dox, multiple iPS lines were generated that could be propagated independently of Dox. As a next step, ‘primary’ iPS lines were injected into blastocysts to generate embryonic or adult mouse chimeras, thus allowing clonal expansion and their re-differentiation into multiple somatic cells types in vivo. Because the injected iPS lines carried a constitutively expressed antibiotic-resistance gene, homogenous iPS-derived somatic cell populations such as embryonic fibroblasts, mesenchymal stem cells, neural precursors and lymphocytes could be isolated that carried identical provirus integration patterns as those in the primary iPS cell line [20,21]. Cultivation of these ‘secondary’ somatic cells in the presence of doxycycline efficiently generated ‘secondary’ iPS cells, which grew independently of Dox and were shown to be pluripotent by stringent criteria. Two major advantages are offered by this strategy. First, because secondary somatic cells do not require new vector-mediated factor transduction, cells that are difficult to infect can be reprogrammed. Second, the approach avoids the genetic heterogeneity produced by direct viral infection of somatic cells [20,21]. More recently, we have also established a secondary system for human cells [24]. By segregating individual proviruses through the germ line, we generated a library of genetically homogeneous mouse embryonic fibroblast lines that carried various combinations of the reprogramming factors, all of which originated from a defined set of proviral genomes [25]. Fibroblast lines lacking any one of the factors were reproducibly competent to generate iPS cells in the presence of doxycycline once the respective missing factor had been re-introduced exogenously. The cell lines and mouse strains described here will be useful for conducting drug screens aimed at replacing the action of individual reprogramming factors.

Reprogramming of somatic cells by a single polycistronic vector

  1. Top of page
  2. Abstract
  3. Introduction
  4. Nuclear transplantation
  5. Fusion of somatic cells and embryonic stem cells
  6. Reprogramming by defined transcription factors
  7. A ‘reprogrammable’ mouse: the ‘secondary’ system
  8. Reprogramming of somatic cells by a single polycistronic vector
  9. Therapeutic potential of iPS cells
  10. Outlook
  11. Acknowledgments
  12. Disclosure of Conflict of Interests
  13. References

Although the generation of induced pluripotent stem cells (iPS) has proven a robust technology in mouse and human, a major impediment to the use of iPS cells for therapeutic purposes has been the viral-based delivery of the reprogramming factors because multiple proviral integrations pose the danger of insertional mutagenesis. We therefore devised a novel approach to reduce the number of viruses necessary to reprogram somatic cells by delivering reprogramming factors in a single virus utilizing 2A ‘self-cleaving’ peptides, which support efficient polycistronic expression from a single promoter. A single provirus transducing the four reprogramming factors Oct4, Sox2, Klf4, and c-Myc was able to generate iPS cells from both embryonic and adult somatic mouse cells [26]. In addition, this approach was successful in generating human iPS lines from human keratinocytes demonstrating that a single polycistronic virus can reprogram human somatic cells.

Therapeutic potential of iPS cells

  1. Top of page
  2. Abstract
  3. Introduction
  4. Nuclear transplantation
  5. Fusion of somatic cells and embryonic stem cells
  6. Reprogramming by defined transcription factors
  7. A ‘reprogrammable’ mouse: the ‘secondary’ system
  8. Reprogramming of somatic cells by a single polycistronic vector
  9. Therapeutic potential of iPS cells
  10. Outlook
  11. Acknowledgments
  12. Disclosure of Conflict of Interests
  13. References

One of the promises of patient-specific ES cells is the potential for customized therapy of diseases. Previous studies have shown that disease-specific ES cells produced by nuclear cloning in combination with gene correction can be used to correct an immunologic disorder in a proof-of-principle experiment in mice [27]. In a similar approach, we recently demonstrated that iPS cells derived from skin cells of a mouse with sickle cell anemia were able to fully restore normal blood function when transplanted into diseased mice [28]. Finally, we have shown that iPS cells can be efficiently differentiated into neural precursor cells giving rise to neuronal and glial cell types in culture. Neural precursors derived from iPS cell were able to improve behavior in a rat model of Parkinson’s disease upon transplantation into the adult brain demonstrating the therapeutic potential of directly reprogrammed fibroblasts for neuronal cell replacement in an animal model [29].

Outlook

  1. Top of page
  2. Abstract
  3. Introduction
  4. Nuclear transplantation
  5. Fusion of somatic cells and embryonic stem cells
  6. Reprogramming by defined transcription factors
  7. A ‘reprogrammable’ mouse: the ‘secondary’ system
  8. Reprogramming of somatic cells by a single polycistronic vector
  9. Therapeutic potential of iPS cells
  10. Outlook
  11. Acknowledgments
  12. Disclosure of Conflict of Interests
  13. References

The understanding of the molecular circuitry that dictates the identity of different cell types has greatly aided our efforts towards reprogramming different somatic cells, including terminally differentiated cells, to a pluripotent state [1]. An unresolved question is whether one somatic cell type can be converted into another cell type without prior dedifferentiation to a pluripotent state, by direct trans-differentiation. Recently, the in vivo conversion of exocrine pancreas cells to endocrine insulin-producing cells has been achieved by expression of three transcription factors [30]. It will be a major challenge for future work to utilize our current knowledge of transcriptional networks active in different somatic cell types to achieve the direct reprogramming of somatic cells to cells of a different germ layer in the Petri dish.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Nuclear transplantation
  5. Fusion of somatic cells and embryonic stem cells
  6. Reprogramming by defined transcription factors
  7. A ‘reprogrammable’ mouse: the ‘secondary’ system
  8. Reprogramming of somatic cells by a single polycistronic vector
  9. Therapeutic potential of iPS cells
  10. Outlook
  11. Acknowledgments
  12. Disclosure of Conflict of Interests
  13. References