Direct conversion of mouse fibroblasts to self-renewing, tripotent neural precursor cells

Abstract

We recently showed that defined sets of transcription factors are sufficient to convert mouse and human fibroblasts directly into cells resembling functional neurons, referred to as "induced neuronal" (iN) cells. For some applications however, it would be desirable to convert fibroblasts into proliferative neural precursor cells (NPCs) instead of neurons. We hypothesized that NPC-like cells may be induced using the same principal approach used for generating iN cells. Toward this goal, we infected mouse embryonic fibroblasts derived from Sox2-EGFP mice with a set of 11 transcription factors highly expressed in NPCs. Twenty-four days after transgene induction, Sox2-EGFP(+) colonies emerged that expressed NPC-specific genes and differentiated into neuronal and astrocytic cells. Using stepwise elimination, we found that Sox2 and FoxG1 are capable of generating clonal self-renewing, bipotent induced NPCs that gave rise to astrocytes and functional neurons. When we added the Pou and Homeobox domain-containing transcription factor Brn2 to Sox2 and FoxG1, we were able to induce tripotent NPCs that could be differentiated not only into neurons and astrocytes but also into oligodendrocytes. The transcription factors FoxG1 and Brn2 alone also were capable of inducing NPC-like cells; however, these cells generated less mature neurons, although they did produce astrocytes and even oligodendrocytes capable of integration into dysmyelinated Shiverer brain. Our data demonstrate that direct lineage reprogramming using target cell-type-specific transcription factors can be used to induce NPC-like cells that potentially could be used for autologous cell transplantation-based therapies in the brain or spinal cord.

Fig. 1.

Induction of a neural precursor-like population with 11 factors. (A) Sox2-EGFP⁺ colonies 25 d after transgene induction by doxycycline. (B) Sox2-EGFP⁺ cells from control (infected only with rtTA) and 11F infections were analyzed by FACS 25 d after transgene induction. (C) qPCR analysis of NPC-specific genes from passage 9 ES cell-derived NPCs (black), passage 4 MEFs (blue), 11F Sox2-EGFP⁺ FACS-sorted cells (red), and negative control rtTA infections (green) 25 d after transgene induction (n = 2). (D) Differentiation of 11F pooled infections 25 d after induction into GFAP⁺ astrocytes (Left) and Tuj1⁺ neurons after 8 d (Right). (Scale bars: 50 mM.)

Fig. 2.

Identification of critical NPC-inducing factors. (A) Quantification of total colonies (black) and Sox2-EGFP⁺ colonies (green) from 11F, two pools 24 d after transgene induction. Control represents infection with rtTA virus alone. Error bars represent SDs of three experiments. (B) Sox2-EGFP⁺ colony 25 d after 5F transgene induction (Left). MAP2⁺ neurons and GFAP⁺ astrocytes were differentiated from the 5F population by removal of mitogens and doxycycline and culturing for 12 d (Right). (C) qPCR analysis of NPC-specific gene induction for Sox2-EGFP⁺ FACS-sorted populations from 5F and 5F−1 pools 25 d after transgene induction (n = 2). (D) When FoxG1 was removed from the 5F pool, no Tuj1⁺ cells with neuronal morphology could be derived. Neuronal cells were observed in all other conditions. Culture conditions are described in B. (Scale bars: 50 μm.)

Fig. 3.

FoxG1+Sox2 can induce a self-renewing bipotent population from fibroblasts. (A) Sox2-EGFP MEF-derived iNPCs give rise to Tuj1⁺ and MAP2⁺ neurons (Upper) and GFAP⁺ astrocytes (Lower Right). EGFP⁺ neuronal cells (Lower Left) can be detected readily from Tau-EGFP MEF-derived iNPCs under the same conditions. Differentiation conditions are described in the text. (B) Sox2-EGFP⁺ cells from FoxG1+Sox2 infections were analyzed by FACS 25 d after infection. (C) Representative current-clamp traces in response to current pulses, in control condition (infection with rtTA virus only; Left) and after infection with FoxG1+ Sox2 (Right). Neurons were differentiated 25 d after transgene induction. Asterisks indicate action potentials. (D) qPCR analysis reveals that FoxG1 and Sox2 can induce a subset of NPC-specific genes but not Brn2 or the region-specific gene Nkx2.2. In addition, the fibroblast-specific gene Col1a1 is not repressed (n = 2). (E) A clonal population derived from the FoxG1+Sox2 condition can differentiate into Tuj1⁺ and MAP2⁺ neurons (Upper) and GFAP⁺ astrocytes (Lower) at multiple passages. (Scale bars: 50 μm.)

Fig. 4.

Addition of Brn2 induces a tripotent NPC population. (A) A Sox2-EGFP⁺ population that gives rise to O4⁺ oligodendrocytes, Tuj1⁺ and MAP2⁺ neurons, and GFAP⁺ astrocytes can be induced from MEFs infected with FoxG1, Sox2, and Brn2. (B) FoxG1 and Brn2 alone induce a population that can give rise to mature CNP⁺, Olig2⁺, and MBP⁺ oligodendrocytes, GFAP⁺ and S100⁺ astrocytes, and TUJ1⁺ and MAP2⁺ neurons. (C) Quantification of TUJ1⁺, GFAP⁺, and CNP⁺ cells from a nonclonal (Left) and clonal (Right) FoxG1+Brn2 iNPC population at multiple passages after 9 d in N3 and 3 d in N2B27 plus 1% serum. (D) Sox2-EGFP⁺ cells from FoxG1+Brn2 infections were analyzed by FACS 21 d after infection. (E) Representative current-clamp traces in response to current pulses. Neurons were differentiated from FoxG1+Sox2+Brn2 (Upper) or FoxG1+Brn2 (Lower) iNPCs 25 d after transgene induction and after 20 passages, respectively. (F) Electrophysiological characterization of FoxG1/Brn2 iNPC-derived and ESC-derived astrocytes. Representative current-clamp traces recorded from iNPC-derived (Left) and ESC-derived (Right) astrocytes in response to current-pulses as in Fig. 3C. (G) FoxG1/Brn2 iNPC-derived astrocytes express the astrocyte-associated markers Aldh1L1, S100B, Aqp4, Igfbp3, and GFAP (n = 2). (H) Passage 17 EGFP-labeled FoxG1/Brn2 iNPCs were transplanted into P1 Shiverer mice targeted to the cerebellum. EGFP⁺ transplanted cells and MBP⁺ myelin tracks were detected 10 wk after transplantation. Right panel shows boxed area in left panel. (Scale bars: 50 μm.)