Efforts to reprogram human somatic differentiated cell types to a state that resembles hESCs began with the pioneering work of Takahashi and Yamanaka11
. Their methods included retroviral integration of four vital reprogramming factors - OCT3/4
, and c-MYC
-into adult human dermal fibroblasts. These four transcription factors would later become known as the “Yamanaka factors”, and their roles in reprogramming are now known to be significant28
but not collectively necessary29–36
. Often the omission of one or more of these reprogramming genes was contingent upon the endogenous network of the donor cell-type. For example, one study found that hiPSC derivation from keratinocytes required only ten days, while neonatal skin fibroblasts required ~30 days37
. It was postulated that perhaps the keratinocytes’ higher endogenous expression levels of c-MYC
predispose them to quicker reprogramming38
. Starting cell-type is thus an important consideration in the derivation process, and a topic that is more thoroughly discussed elsewhere39
. Two other transcription factors, namely NANOG
, were initially shown to be able to substitute for c-MYC
, although a number of other different factor combinations have been subsequently demonstrated26, 29, 36, 40, 41
. In any event, several cocktails comprising any number of these six reprogramming factors, and in some cases, additional supplements such as small molecules and enzymes, have been shown to be capable of reprogramming cells to pluripotency.
A chief aim of clinical hiPSC researchers is to achieve a high efficiency of derivation of hiPSCs, as current yields of bona fide hiPSCs can be as low as 0.001-0.1% of the starting cell population42, 43
. Even in so-called “secondary” reprogramming systems, in which all of the somatic cells homogeneously express the reprogramming factors, the efficiency of inducing pluripotency remains low at 1–5%. Two mutually non-exclusive models have been proposed to explain the apparent resistance to pluripotency induction, termed the “elite” and “stochastic” models44
. The elite model proposes that only a small percentage of somatic cells, presumably resident tissue progenitor cells, are amenable to reprogramming. In support of this notion is evidence that hematopoietic stem cells undergo more efficient reprogramming than their differentiated progeny45
. However, reports of successful reprogramming of terminally differentiated cells such B lymphocytes46
and pancreatic β islets47
favor a stochastic model of reprogramming, in which successive cell divisions allow rare cells to acquire the stochastic changes necessary for conversion to full pluripotency48
. Perhaps these seemingly contradictory hypotheses can be reconciled by a model in which adult stem/progenitor cells require fewer stochastic changes to undergo reprogramming than more differentiated cells. Further investigation of the reprogramming process using single-cell resolution imaging and other techniques will undoubtedly help yield further insight into these reprogramming roadblocks.
Clearly, the choice of gene delivery vector can change reprogramming efficiency by directly affecting the degree of expression of the reprogramming genes. Retroviral/lentiviral infection provides the benefit of high transgene expression levels in primary cells as compared to nonviral methods of reprogramming. However, retroviral/lentiviral methods for hiPSC generation have come under scrutiny due to concerns regarding their ultimate clinical safety. In particular, the random integration of transgenes into the human genome can potentially cause insertional mutagenesis, leading to malignant transformation of a clonal cell population and disastrous consequences49
. Second, leaky expression due to ineffective silencing of the transgenes may interfere with the physiological expression of the factors endogenously present within the cell, thereby potentially restricting differentiation propensity50
. This residual expression may hamper the validity of in vitro
hiPSC uses, such as in disease modeling, drug screening, and toxicology tests. Third, reactivation of OCT4
has been shown to promote tumor formation in chimeric mice26, 51, 52
, prompting legitimate concern over post-transplantation tumorigenic risk if such methods were employed in human patients.
In one of the initial forays into generating safer hiPSCs, Maherali et al. created a doxycycline-inducible lentiviral system, attempting to maintain the silencing of transcription factors and thus reduce the tumorigenicity of the cells post-differentiation37
. Although this system is a step towards safer hiPSCs, the leakiness of the doxycycline-inducibile promoter and the permanent incorporation of oncogenes into the host genome still warrant concern.
Viral integration followed by excision: Cre-loxP
Soldner et al. generated viable hiPSCs free of exogenous reprogramming factors using doxycycline-inducible lentiviral vectors that integrated into the host genome, but were subsequently excised by Cre recombinase53
. Fibroblasts were obtained via skin biopsies from five patients exhibiting sporadic Parkinson’s Disease, and transduced using three or all of the Yamanaka factors. The reported reprogramming efficiency was 0.005% after transduction with the three-factor combination and 0.01% with the four-factor combination. Furthermore, the three-factor transduced cells required twelve days of DOX-exposure, as opposed to the four-factor cells, which required only eight days. Despite its lower reprogramming efficiency and temporal requirements, the three-factor may be preferred over the four-factor combination since the transduced cells are not overgrown by granulate colonies11, 31
. Southern blot analysis demonstrated successful excision of the transgenes, and the resulting hiPSC lines maintained pluripotency independent of residual exogenous transcription factor expression.
Since the isolated hiPSC lines are patient-specific, they provide a system for investigating the proposed molecular and cellular mechanisms of the disease. Soldner et al. demonstrated successful derivation of dopaminergic neurons from Parkinson’s Disease patients’ cells, indicating that the underlying age or disease of the donor most likely does not affect the ability of their cells to produce hiPSC-derived replacements ex vivo53
. Interestingly, in this study, factor-free hiPSCs were found to be more closely related to embryo-derived hESCs than provirus-carrying parental hiPSCs based on gene expression analysis. Hence, basal expression of proviruses carried in conventional hiPSCs can affect the molecular characteristics of the hiPSCs. Although transgenes are expected to be completely silenced in bona fide hiPSC lines, residual sequences and chromosomal disruptions during and after viral integration may still result in harmful alterations that could pose clinical risks. As previously mentioned, reactivation of reprogramming transgenes post-transplant can result in malignant transformation of the cells and formation of a tumor15
. Such unpredictable effects of incomplete transgene silencing on downstream hiPSC phenotype further highlight the need for transgene-free hiPSC derivation methods.
Nonviral integration followed by excision: piggyBac Transposition
Although Cre recombinase-driven excision utilizes a highly efficient and widely-used system, small residual vector backbone sequences remain at the site of integration and may engender unpredictable downstream effects. Woltjen et al. and Kaji et al. demonstrated successful reprogramming of human embryonic fibroblasts using doxcycline-inducible reprogramming factors that were delivered as plasmids, stably integrated into the host genome, and subsequently excised using piggyBac transposition54, 55
. Woltjen et al. noted that successful transposon-based nonviral reprogramming has several advantages over traditional lentiviral integration-based reprogramming: (1) improved accessibility of reprogramming techniques through the use of plasmid DNA preparations and commercial transfection products, thereby eliminating the need for specialized biohazard containment facilities; (2) increased variety of reprogrammable donor cell-types because susceptibility to viral infection is no longer an important consideration; (3) feasibility of xeno-free production of hiPSCs; and (4) most importantly, near complete elimination of the expression of reprogramming factors after establishment of hiPSC lines by piggyBac transposase-mediated excision. However, successful excision of the reprogramming cassette was only achieved in approximately 2% of the bona fide hiPSCs exposed to piggyBac transposase, limiting the amount of vector-free hiPSCs that could potentially be produced. Of note, Mali et al. demonstrated the use of butyrate to enhance reprogramming efficiency 15- to 51-fold when used in conjunction with piggyBac transposase-driven integration and excision of the Yamanaka reprogramming factors56
. Genome-wide analysis of the effects of butyrate exposure at days 6–12 demonstrated significant changes in H3 acetylation and promoter methylation status in a variety of pluripotency-related genes, including DPPA2.