A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types

Abstract

The study of induced pluripotency is complicated by the need for infection with high-titer retroviral vectors, which results in genetically heterogeneous cell populations. We generated genetically homogeneous 'secondary' somatic cells that carry the reprogramming factors as defined doxycycline (dox)-inducible transgenes. These cells were produced by infecting fibroblasts with dox-inducible lentiviruses, reprogramming by dox addition, selecting induced pluripotent stem cells and producing chimeric mice. Cells derived from these chimeras reprogram upon dox exposure without the need for viral infection with efficiencies 25- to 50-fold greater than those observed using direct infection and drug selection for pluripotency marker reactivation. We demonstrate that (i) various induction levels of the reprogramming factors can induce pluripotency, (ii) the duration of transgene activity directly correlates with reprogramming efficiency, (iii) cells from many somatic tissues can be reprogrammed and (iv) different cell types require different induction levels. This system facilitates the characterization of reprogramming and provides a tool for genetic or chemical screens to enhance reprogramming.

Figure 1. Generation of genetically homogeneous cell cultures for epigenetic reprogramming

(a) Scheme for infection of puromycin-resistant, Nanog-GFP or Nanog-neo primary MEFs expressing the reverse tetracycline transactivator (M2rtTA) with dox-inducible lentiviruses encoding the four reprogramming factors followed by induction of reprogramming, primary iPS cell colony selection, dox withdrawal, chimera formation and puromycin selection for iPS cell–derived secondary somatic cells. (b) NNeo secondary MEFs isolated from chimeras undergo complete epigenetic reprogramming. Dox-independent cultures express the pluripotency-associated genes alkaline phosphatase, SSEA1 and Nanog. (c,d) MEF-derived NNeo and NGFP2 secondary iPS cells generate cells of all three germ layers in teratomas (c; arrow, mesoderm; asterisk, endoderm; cross, ectoderm) produced in teratoma formation assays and contribute to chimera formation when injected into blastocysts, as indicated by the presence of iPS cell–derived agouti coat color on a black background (d). Scale bars, 250 µm.

Figure 2. Reprogramming kinetics and efficiencies vary between MEFs from distinct iPS cell lines

(a) Secondary MEFs from three ‘primary’ iPS cell lines were treated with dox, and reprogramming was monitored visually. The different MEF populations exhibited morphologic differences 6 d after dox administration, but all formed colonies with ES-cell morphology within 12 d (arrows). Scale bar, 250 µm. (b) Neo-resistant and alkaline phosphate–positive colonies were present in NNeo cultures when the drug was added to the media as early as day 4 after dox induction. (c) Flow cytometric analysis for reactivation of SSEA1 and the Nanog-GFP reporter allele (in NGFP2 and NGFP3 lines) over 18 d of dox culture. (d) Secondary NGFP2 MEFs were plated at densities varying from 0.025–500 cells/mm² followed by dox addition. GFP⁺ colonies were counted 4 weeks later. (e) Single secondary MEFs were plated in 96-well plates containing a γ-irradiated MEF feeder layer followed by dox induction. The percentage of single cells able to proliferate sufficiently to form a visible colony on the MEF feeder layer (light gray bars) and the percentage of single cells able to form GFP⁺ or neo-resistant secondary iPS cell colonies (dark gray bars) were scored 4 weeks later. (f) Comparison of the interexperimental variability in iPS cell colony formation efficiency between direct infection and the secondary system. 3 × 10⁵ Oct4-neo MEFs derived from a single embryo were infected with the four factors encoded by Moloney-based retroviral vectors on a 10-cm plate, neo selection was initiated on day 6 and resistant colonies were counted on day 20 (left, direct infection). 3 × 10⁴ secondary NGFP2 MEFs derived from one chimeric embryo were plated in a six-well dish, exposed to dox-containing media and GFP⁺ colonies were counted 3 weeks later (right, secondary system). The bars represent number of colonies in each of the four independent experiments.

Figure 3. Requirement and expression of four-factor transgenes in secondary MEFs

(a) Quantitative RT-PCR examining induction of expression of the four reprogramming factors in response to 72 h of dox treatment, relative to GAPDH levels. Error bars represent s.d. (n = 3). (b) Immunofluorescence detecting Oct4 and Sox2 in secondary MEF cultures 72 h after dox induction. Scale bar, 50 µm. (c) NGFP2 secondary MEFs were cultured in the presence of dox for the indicated time (5–22 d, red bars) followed by dox withdrawal. Cultures were monitored daily for the first instance of GFP activation (green bars). Blue bars indicate periods in which GFP⁺ colonies appeared during dox treatment. (d) NGFP2 MEFs were cultured in the presence of dox for 10–15 d, at which point dox was withdrawn; GFP⁺ colonies were scored at day 34. (e) NGFP2 MEFs were cultured in the presence of dox for either 9 (blue) or 22 d (red line), and the appearance of GFP⁺ colonies was scored daily until day 29. Note the appearance of GFP⁺ colonies as late as 15 d after dox withdrawal (blue line). (f) NGFP3 secondary MEFs were cultured in the presence or absence of dox, dox + 5-Aza, or dox + TSA, and GFP⁺ colonies were scored 3 weeks later. Error bars represent s.d. (n = 3).

Figure 4. Reprogramming of intestinal epithelial cells

(a) NNeo secondary intestinal epithelial crypt-villus structures were isolated from chimeras. (b) After 24 h of culture in the presence of dox, spheroids began appearing in suspension (inset). (c) Within 72 h of dox culture, suspended spheroids attached to the γ-irradiated feeder layer and acquired an ES cell–like morphology. (d,e) Colonies continued to grow during 2 weeks of dox treatment (d), but differentiated and became indistinguishable from the feeder layer upon dox withdrawal (e). (f) Dox-dependent intestinal epithelial colonies were neo resistant 2 weeks after dox administration. Scale bars (a–f), 250 µm. (g) Bisulfite sequencing of the endogenous Oct4 and Nanog promoters in freshly isolated NNeo secondary intestinal epithelium, partially reprogrammed dox-dependent cells, fully reprogrammed NNeo iPS cells after infection with Sox2 and Klf4 viruses. (h) Quantitative RT-PCR analyses of expression of the four factors and Nanog revealed that dox-dependent NNeo intestinal epithelial (IE) colonies express Oct4 and c-Myc at high levels compared with ES cells, but Sox2 and Klf4 at very low levels. (i) NGFP2 secondary intestinal epithelial cells also formed spheroids in suspension within 24 h of dox addition and acquired an ES cell–like morphology within 72 h (j). (k,l) NGFP2 intestinal epithelium gave rise to dox-independent secondary iPS cell colonies that express GFP from the endogenous Nanog locus. Scale bars (i,l), 50 µm. (m) EDTA-DTT–based fractionation of intestinal villi from differentiated cells of the tip (fraction 1) to the progenitor cells of the crypt (fraction 7) followed by 4 d of dox induction demonstrates that crypt fractions in both NNeo and NGFP2 secondary lines are more efficient at initial colony formation. (n) Quantitative RT-PCR analysis showed that with the exception of Klf4, the transgenes were more efficiently induced in fraction 7 (crypt) than in fraction 1 (villus tip) of the NNeo and NGFP2 intestinal epithelial cells.

Figure 5. Reprogramming of other somatic cell types

(a–b) NNeo mesenchymal stem cells (MSCs) before and after 3 weeks of dox administration. (c–d) NGFP2 MSCs before and after 10 d of dox treatment forming ES cell–like colonies. (e–f) NGFP2 MSCs gave rise to dox-independent iPS cell colonies that express GFP from the endogenous Nanog locus. (g,h) Colonies of dermal keratinocytes from NNeo chimeras with typical epithelial morphology (inset) (g) began to exhibit ES-cell morphology within 12 d of dox treatment (h). (i) These cells fully reprogrammed to form neo-resistant secondary iPS cell colonies. (j) After expansion in serum-free media, plated NNeo-derived neurospheres readily differentiated into astrocytic cells in response to dox- and serum-containing ES cell media. (k,l) When plated neurosphere cells were expanded in adherent conditions with EGF and FGF2 for another 3 weeks and then exposed to dox-containing media, iPS cell–like colonies appeared both in ES cell (k) and serum-free media (l). Scale bars (a–d) and (g–j), 250 µm; (e–f) and (k–l), 50 µm.