Abstract

Results

Our experiments initially focused on the question of whether mRNA reprogramming could be enhanced using engineered variants of Oct4 incorporating an N-terminal MyoD transactivation domain^20,21 or a C-terminal triple repeat of the VP16 transactivation domain²², or by augmenting the cocktail with transcripts for two additional factors, Rarg and Lrh-1²³. To this end, we carried out a test to compare the performance of six different mRNA cocktails comprising transcripts for the five RFs currently used in mRNA reprogramming (Oct4, Sox2, Klf4, c-Myc-T58A and Lin28), or a 7-factor cocktail including Rarg and Lrh-1, each combination being tried in three variations based on wild-type Oct4 and the MyoD- and VP16-Oct4 fusion constructs (designated M₃O and VPx3, respectively). BJ fibroblasts plated at 10K cells per well were transfected in feeder-based cultures for 11 days, by which time advanced morphologies were apparent in several of the wells. Over the next few days colonies with characteristic human embryonic stem cell (hESC) morphology emerged in wells transfected with the cocktails based on wild-type Oct4 and M₃O, while the target cells in the VPx3-transfected cultures retained a fibroblastic morphology—albeit while showing accelerated growth and some tendency to aggregate into foci—and no colonies emerged (data not shown). The 5-factor and 7-factor versions of the wild-type Oct4 and M₃O-based cocktails demonstrated similar colony productivity, hence no advantage accrued from the inclusion of Rarg and Lrh-1. However, the M₃O cocktail gave several times the number of colonies produced with wild-type Oct4. Colonies were picked from the M₃O 5-factor well for expansion and further analysis (Fig. 1). Pluripotency of M₃O-derived colonies was confirmed by staining for nuclear and cell-surface markers (Fig. 1b), and by in vitro differentiation into the three primary germ layers (Fig. 1c–e and Supplementary Movie 1). Six expanded clones were subjected to karyotype analysis and DNA fingerprinting, karyotypic normality and BJ lineage being confirmed in all cases (data not shown).

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Figure 1

iPSC Colonies Derived with M₃O-Based mRNA Reprogramming Cocktail.

(a) Phase contrast imagery showing morphology of an iPSC colony picked from the first M₃O-based BJ reprogramming plate and replated onto feeders. (b) Immunostaining of an expanded clone revealing expression of cell-surface and nuclear pluripotency markers. (c) Immunofluorescence images showing expression of neuro-ectoderm markers after in vitro differentiation of the iPSC clone shown in the previous panel. (d) Immunostaining showing expression of the endodermal marker Sox17 in cells derived from the same clone subjected to in vitro endodermal differentiation. (e) α-Actinin immunostaining of cardiomyocytes derived by mesodermal differentiation of the same iPSC clone (see also Supplementary Movie 1).

In a follow-up experiment we investigated whether the potency of the mRNA cocktail could be further enhanced by the inclusion of Nanog transcripts²⁴. Four wells containing 50K BJ fibroblasts on feeders were transfected with wild-type Oct4 or M₃O-based 5-factor or 6-factor cocktails for six days, and each culture was then passaged 1:6 onto fresh feeders to populate a 6-well plate (Fig. 2a). Transfection was continued for up to 5 more days within each plate. Cultures were fixed and stained with TRA-1-60 antibodies on day 18 (where day 0 corresponds to the first transfection) to assess the impact of the different cocktails and transfection time courses on iPSC productivity (Fig. 2b–c). The results showed that adding Nanog to the cocktail was highly beneficial regardless of the Oct4 variant employed, while the greatest conversion efficiency was attained when M₃O and Nanog were used together.

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Figure 2

Comparison of Reprogramming Efficiency Using 4 Different mRNA Cocktails.

(a) Flowchart summarizing the feeder-based 4-cocktail comparison experiment described in the text. The 5-factor reprogramming cocktails comprised wild-type Oct4 or M₃O plus Sox2, Klf4, c-Myc-T58A, and Lin28 mRNA transcripts, while 6-factor cocktails included these RFs plus Nanog. (b) Immunofluorescence imaging showing the yield of TRA-1-60⁺ colonies produced by the four different cocktails at day 18 of the experiment. The total number of days of transfection is indicated for each culture. (c) Chart showing the yield of TRA-1-60⁺ colonies at day 18 versus the number of days of transfection using the four cocktails.

We confirmed the efficacy of the M₃O-based 5-factor or 6-factor cocktails in additional feeder-based experiments using three additional human fibroblast lines (data not shown). Reprogramming kinetics and efficiency were improved with these low-passage lines (HDF-f, HDF-n and XFF), which displayed faster population doubling times than the relatively late-passage BJ cells in expansion culture. In some cases, hESC-like colonies were obtained following as little as six days of transfection, although the yields were higher when transfection was continued for a few more days. These experiments did reveal a drawback to the M₃O-based cocktail in that attrition of the mitotically-arrested feeder cells was markedly accelerated when using the engineered Oct4 (data not shown). The rapid loss of the feeder layer appeared to have an adverse impact on the survival and proliferation of the target cells, suggesting that the performance advantage from using M₃O over wild-type Oct4 might be heightened if we could either find a way to sustain the feeder layer or switch to a feeder-independent system. Experiments involving periodic reinforcement of the feeder layer by the addition of fresh cells suggested that while such a strategy might be of some benefit, this would be offset by the added complexity of the protocol (data not shown). We therefore decided to focus on applying the more potent cocktail to establish a streamlined, feeder-free protocol.

Feeder-independent iPSC derivation has generally proved somewhat challenging regardless of the reprogramming technology employed, but raises special difficulties in the context of a sustained transfection regimen. There is a lower limit to the density at which fibroblasts can be plated without compromising cell viability and proliferative activity²⁵. The propensity of cells to undergo mitotic arrest or apoptosis in sparse cultures is exacerbated when they are stressed by transfection and by ectopic expression of reprogramming factors. Moreover, RNA doses which are well tolerated at the high cell densities characteristic of feeder-based reprogramming produce more severe cytotoxic effects when distributed among fewer cells. At the same time, the penetrance of expression achieved with mRNA transfection declines after fibroblasts reach confluence, perhaps due to down-regulation of endocytosis associated with contact inhibition and cell-cycle arrest. Reduced permissiveness to transfection in crowded cultures tends to be alleviated during mRNA reprogramming once cells undergo mesenchymal-to-epithelial transition (MET). However, the ~7 days required for human fibroblasts to reach MET using current mRNA cocktails renders it difficult to avoid fibroblastic overgrowth even when cells are plated at the lowest survivable initial densities. Cultures can be thinned by passaging to postpone this fate, but the plating efficiency of highly stressed reprogramming intermediates is unpredictable, and a split-based protocol would in any case sacrifice convenience and scale poorly to high-throughput applications.

Phenotypic indications of MET (involution of fibroblastic processes and the emergence of foci and cobblestone morphologies) were accelerated in experiments with enhanced cocktails, so we attempted feeder-free derivations using the 6-factor M₃O cocktail without passaging steps starting with fibroblast cultures seeded at from 50K to 150K cells per well. The fate of the reprogramming cultures proved highly sensitive to the initial seeding density, presumably because the deleterious effects of excessive cytotoxicity and fibroblastic overgrowth compound over the course of days of transfection. In an early experiment employing a uniform RNA dosing regimen, dozens of hESC-like colonies were obtained from neonatal HDF-n and XFF fibroblasts plated at 100K per well, while the population density in 50K wells crashed and 150K wells succumbed to fibroblastic overgrowth, these sub-optimal wells produced only a few colonies each (data not shown). When the same protocol was applied to two other fibroblast lines, BJ and HDF-a (an adult-derived line), even the most promising (150K) cultures became largely quiescent shortly after reaching confluence, subsequently yielding only sporadic colonies with delayed kinetics. In our follow-on experiments we switched from ambient to 5% oxygen culture and ramped to full RNA dosing from a quarter dose over the first four days of transfection, in efforts to minimize the impact of stress-induced cellular senescence^{26,27,28,29,30,31}. In the first trial using these new conditions we tested if substitution of wild-type L-Myc for c-Myc-T58A could further improve reprogramming efficiency³², and in parallel evaluated a simplified transfection scheme by which RNA was added to cells with daily media changes rather than being delivered in a separate step four hours earlier. RNA dosing was scaled down in 24-hour transfection wells to compensate for an expected increase in cytotoxicity, and two different transfection reagents were tested with this regimen (RNAiMAX and Stemfect). XFF cells were plated at 50K per well and transfected for 9 days. Productivity was assessed by counting TRA-1-60⁺ colonies at the experiment end-point (Fig. 3a–b). The more conventional 4-hour transfection regime yielded about 250 TRA-1-60⁺ colonies per well by day 15 using the c-Myc-based cocktail, equivalent to a reprogramming efficiency of 0.5% (CV = 21%, n = 3), while the corresponding L-Myc cultures displayed an average reprogramming efficiency of only ~0.02%. Reprogramming kinetics and efficiency were improved across the board with the 24-hour transfection regime, and these cultures were fixed for analysis on day 11 to avoid overgrowth in the most productive wells. Using the c-Myc cocktail, the RNAiMAX condition gave on the order of a thousand colonies per well at all three RNA dosages tested, corresponding to an iPSC conversion efficiency of ~2%. The yield of TRA-1-60⁺ colonies was still higher using the Stemfect reagent, making it impossible to count colonies in the most productive wells. In the best-performing c-Myc 400 ng/ml condition, the culture was almost completely overrun with hESC-like cells as early as day 9, one day after the last transfection (Fig. 3c). The mechanistic basis for the superior performance of the 24-hour regimen is unknown, but it may be relevant that dose ramping was achieved within these wells by delivering a reduced volume of medium containing a fixed concentration of RNA, and this might have increased the effective density of thinly-plated cultures. When the preferred conditions were applied in a follow-up derivation on HDF-a adult-derived fibroblasts, 50K and 100K cultures gave a handful of colonies each, but wells seeded with 75K cells produced abundant hESC-like colonies with similar kinetics to the highly proliferative XFF cells (Fig 3d). The rapid kinetics of colony formation using these conditions is apparent in time–lapse imaging showing a subsequent XFF-based derivation presented in Supplementary Movie 2.

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Figure 3

Feeder-Free Reprogramming Using M₃O-Based Cocktail.

(a) Immunofluorescence imaging showing the TRA-1-60⁺ colony yield from feeder-free derivations on XFF fibroblasts seeded at 50K per well, comparing c-Myc and L-Myc-based cocktails and 4-hour and 24-hour transfection regimens. All wells were transfected for 9 days. The 4-hour transfection cultures were fixed for staining on day 15 of the experiment, and the 24-hour transfection cultures on day 11. Final media concentration of the transfected RNA is indicated for 24-hour transfection wells. (b) Bar charts showing the TRA-1-60⁺ colony counts for the wells in the preceding panel. Note that the bulk conversion achieved in the most productive Stemfect wells rendered it impossible to distinguish and count individual iPSC colonies. (c) Phase image of the 400 ng/ml c-Myc Stemfect well from the experiment showing hESC-like colonies overtaking the culture on day 9 of the derivation. (d) Phase imagery showing emergence of colonies from HDF-a adult fibroblast cultures seeded at 75K cells per well and subjected to the same 9-day 400 ng/ml Stemfect transfection regime.

Discussion

We have doubtless only scratched the surface in terms of the potential for technical improvements to RNA-based reprogramming. Size-based purification of synthetic mRNA may further reduce activation of cellular antiviral responses and any associated priming of senescence pathways, while possibly obviating the need for an interferon inhibitor^33,34,35. Numerous candidate RFs have been identified which might yield additional performance gains if incorporated into mRNA cocktails^{36,37,38,39,40}, while the advantage realized by substituting M₃O for wild-type Oct4 offers fresh support for the application of engineered chimeric factors to cell-fate direction. Acceleration of the reprogramming process could likewise come about through optimization of factor stoichiometries⁴¹, and we note that the mRNA method opens up the possibility of fine-tuning cocktails to address early and late phases of iPSC induction independently. Finally, the reagents and approach applied here for sustained delivery of mRNA can also be employed to co-transfect other types of RNA including miRNA, which has proven utility for iPSC generation^42,43,44. The methodology we describe already represents a major advance over current protocols. It cuts mRNA reprogramming time by as much as a half with even greater reductions in hands-on time and material costs, while removing troublesome steps from the procedure and allowing mRNA to be handled as a media supplement with almost the same facility as growth factors and cytokines. At the same time, the feeder-free protocol meets a key requirement for moving iPSCs from laboratory to clinic by eliminating potential sources of non-human contamination from the derivation process. If this fast, economical footprint-free iPSC origination technique can be combined with advances in high-throughput propagation and quality assurance of pluripotent clones⁴⁵, the remaining barriers to large-scale, cost-effective production of therapeutically-qualified patient-specific iPSC lines should be few indeed.

Methods

Author Contributions

LW and JW planned the project. YN and XG participated in discussion. XG provided funding and support. LW conducted the majority of the experiments. JW and YN performed certain plasmid construction work. LW wrote the manuscript; YN helped with the videos; JW finalized the paper.

Supplementary Material

Acknowledgments

Footnotes

References