Direct reprogramming of mouse fibroblasts to neural progenitors

Abstract

The simple yet powerful technique of induced pluripotency may eventually supply a wide range of differentiated cells for cell therapy and drug development. However, making the appropriate cells via induced pluripotent stem cells (iPSCs) requires reprogramming of somatic cells and subsequent redifferentiation. Given how arduous and lengthy this process can be, we sought to determine whether it might be possible to convert somatic cells into lineage-specific stem/progenitor cells of another germ layer in one step, bypassing the intermediate pluripotent stage. Here we show that transient induction of the four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) can efficiently transdifferentiate fibroblasts into functional neural stem/progenitor cells (NPCs) with appropriate signaling inputs. Compared with induced neurons (or iN cells, which are directly converted from fibroblasts), transdifferentiated NPCs have the distinct advantage of being expandable in vitro and retaining the ability to give rise to multiple neuronal subtypes and glial cells. Our results provide a unique paradigm for iPSC-factor-based reprogramming by demonstrating that it can be readily modified to serve as a general platform for transdifferentiation.

Fig. 1.

Transdifferentiation by transient expression of the conventional four reprogramming factors generates functional neural stem/progenitor cells. (A) Scheme for the transdifferentiation of dox-inducible secondary MEF cells into neural stem/progenitor cells (NPCs). Duration of dox (4 μg/mL) treatment is for the indicated number of days. Different media were added sequentially as described in Materials and Methods. (B) Pax6 immunostaining on day 13 of colonies arising from the indicated durations of dox treatment; 8 μg/mL dox was used for the 3-d treatment. (C) Number of PLZF-expressing colonies generated with different durations of dox treatment, as analyzed on day 13. Percentages of PLZF⁺ colonies over total colonies are shown. (D) Immunostaining of colonies on day 13 with various neural or neuronal markers. (E) Immunostaining of spontaneously differentiated cells from isolated colonies on day 13 with various mature neuronal or glial markers. Long-term differentiated neurons showed characteristic synapsin I expression patterns (F) and generated a full train of action potentials during injection of 20 pA current (whole-cell configuration in the current-clamp mode) (G) as well as fast Na⁺ currents and outward K⁺ currents (whole-cell configuration in the voltage-clamp mode) (H). Mature neurons also showed glutamate-mediated excitatory postsynaptic currents (EPSCs, I, i), indicating synapse formation between neurons. EPSC amplitude was partially blocked by the AMPA-type receptor antagonist NBQX (I, ii), and the remaining component was completely abrogated by the NMDA-type receptor antagonist APV (I, iii) (whole-cell configuration in the voltage-clamp mode). (Scale bars, 100 μm.)

Fig. 2.

Direct reprogramming is a highly efficient method of deriving pure neural progenitor cells. (A) Experimental overview and cartoon representation of results from panels B, D, and F. Cells after 9 d of differentiation from iPSCs, or after 13 d of transdifferentiation show the indicated marker expression profiles. (B) Day 9 immunostaining of cells differentiated from iPSCs, or transdifferentiated NPCs on day 13, with antibodies against the indicated markers. Pax6 and Sox1 demarcate early neuroectoderm. Sox17 is indicative of endoderm. T expression is early mesodermal. (Scale bars, 100 μm.) (C–F) qRT-PCR analysis of the indicated markers’ expression in cells harvested at multiple time points during the direct reprogramming process (C and D) and differentiation from iPSCs (E and F). Pluripotency genes (C and E) and lineage-specific genes (D and F) are shown in separate graphs. All values are relative to expression in iPSCs.

Fig. 3.

Histone modification during direct reprogramming. Sox1 and Oct4 promoter loci were analyzed by chromatin immunoprecipitation with antibodies against the epigenetic marks of H3K4me3 and H3K27me3 during direct reprogramming. Samples were obtained on day 0 (D0), day 4 (D4), day 8 (D8), and day 12 (D12). Adult NPCs (aNPC) were used as a control. Blue and red bars indicate the extent of immuoprecipitation from normal IgG and each of the antimodified histone antibodies, respectively. Data are percentage of input chromatin amount, analyzed by qPCR (mean ± SEM, n = 3).

Fig. 4.

Fate choice is dictated early during the transdifferentiation process by different environmental cues. (A) Schematic of experiments involving differential exposure to LIF- containing medium (RepM-Pluri) from day 4 onward for the indicated number of days. Dox treatment was for 5 d, beginning on D0. (B) PLZF⁺ colony number expressed as a percentage of the total from each indicated sample on day 9. (C and D) Quantitative analysis of mRNA levels of indicated marker genes in each sample (harvested on day 9). All values are relative to expression in iPSCs. Pax6 immunostaining (E) and total colony number (F) of TTFs transdifferentiated in the presence or absence of JAK inhibitor. Immunostaining of reprogrammed NPCs from TTFs; neural rosette formation (G) and their long-term differentiation into various neuronal subtypes (H–L). (Scale bars, 100 μm.)

Fig. 5.

Model for direct reprogramming of fibroblasts (Fib.) to neural stem/progenitor cells. By adding neural medium to four factor-induced intermediate cells comprising various epigenetic states, a fate switch to neural stem/progenitor cells (NPCs) can be achieved. Alternatively, iPSCs can be generated by prolonged expression of the four factors with concomitant incubation in ESC medium. Cells belonging to other lineages could likely also be isolated, depending on the type of medium used.