CEND1 and NEUROGENIN2 Reprogram Mouse Astrocytes and Embryonic Fibroblasts to Induced Neural Precursors and Differentiated Neurons

Abstract

Recent studies demonstrate that astroglia from non-neurogenic brain regions can be reprogrammed into functional neurons through forced expression of neurogenic factors. Here we explored the effect of CEND1 and NEUROG2 on reprogramming of mouse cortical astrocytes and embryonic fibroblasts. Forced expression of CEND1, NEUROG2, or both resulted in acquisition of induced neuronal cells expressing subtype-specific markers, while long-term live-cell imaging highlighted the existence of two different modes of neuronal trans-differentiation. Of note, a subpopulation of CEND1 and NEUROG2 double-transduced astrocytes formed spheres exhibiting neural stem cell properties. mRNA and protein expression studies revealed a reciprocal feedback loop existing between the two molecules, while knockdown of endogenous CEND1 demonstrated that it is a key mediator of NEUROG2-driven neuronal reprogramming. Our data suggest that common reprogramming mechanisms exist driving the conversion of lineage-distant somatic cell types to neurons and reveal a critical role for CEND1 in NEUROG2-driven astrocytic reprogramming.

Figure 1

CEND1 and/or NEUROG2 Overexpression Drives Astrocytes toward Radial Glia Phenotype (Top) Schematic drawing of the protocol used for astrocytic reprogramming using retroviral vectors overexpressing the neurogenic factors CEND1 and NEUROG2. (A–H) The vast majority of the cells in control GFP-virus-transduced cultures were positive for glial fibrillary acidic protein (GFAP) (A and E). Overexpression of CEND1 (B and F), NEUROG2 (C and D), or both (D and H) resulted in a decrease in the number of GFAP⁺ astrocytes (B and C) and an important increase of GFAP⁻ cells with elongated morphology that strongly expressed the radial glia marker GLAST (F–H). (I–L) Quantification of at least three independent experiments showed different cell types present in control astrocytic cultures (I) and CEND1 (J), NEUROG2 (K), and double-transduced (L) cultures on the basis of their morphology and expression of GLAST and GFAP and revealed the decrease in the astrocytic population with parallel increase of radial glia population upon CEND1 and/or NEUROG2 overexpression. (L) In double-transduced cultures in particular, a new cell population of small round cells appeared, which 48 hr after transduction increased up to 20%. (M–O) 48 hr after CEND1 and/or NEUROG2 transduction, βIII-TUBULIN⁺ cells appeared in the astrocytic cultures only in cells overexpressing CEND1 (M), NEUROG2 (N), or both (O). Nuclei in (A) and (N) were stained by TOPRO-3 (blue). Scale bars, 40 μm. See also Figure S1.

Figure 2

Distinct Subtype-Specific Neuronal Identity Acquired by Reprogrammed Astrocytes following CEND1 and/or NEUROG2 Forced Expression (A–K) Differentiated neuronal morphology, subtype-specific neuronal phenotype, and synaptic marker expression in neurons derived from reprogrammed astrocytes after 20 days in culture, following CEND1 (A–D), NEUROG2 (E–G), and CEND1 and NEUROG2 (H–K) overexpression. CEND1 overexpression directed reprogrammed neurons (A) toward acquisition of GABA⁺ identity (B), whereas NEUROG2 overexpression directed reprogrammed neurons toward TH⁺ (G) and glut⁺ (E) identity. Both GABA⁺ and TH⁺ cells were present upon forced expression of both molecules (H–I). Functional markers PSD95, indicating post-synaptic activity (D, F, and J, arrows), and SYNAPSIN, indicating pre-synaptic activity (C, G, and K, arrows), were present in the synaptic membranes of reprogrammed NEUN⁺ and DOUBLECORTIN⁺ neurons. (L) βIII-TUBULIN⁺ cells appeared in astrocytic cultures 48 hr after CEND1 and/or NEUROG2 overexpression, and their percentage significantly increased after 72 hr. (M) Percentages of GABA⁺ and TH⁺ neurons in the βIII-TUBULIN⁺ neuronal population 72 hr after CEND1 and/or NEUROG2 forced expression. DCX, DOUBLECORTIN. Nuclei were stained by TOPRO-3 (blue). Data were collected from at least three independent experiments. Scale bars, 40 μm. See also Figure S2.

Figure 3

Long-Term Live-Cell Imaging upon CEND1 Overexpression (A–L) Astrocytic cultures transduced with the CEND1-IRES-GFP retrovirus trans-differentiated, giving rise to βIII-TUBULIN⁺ neurons (G) after passing through one or two divisions (lineage trees H to L). Snapshots of the following time points during 1 week live-cell imaging: 5 min (A), 41 hr (B), 66 hr (C), 100 hr (D), 125 hr (E), and 155 hr (F). Cell tracking and lineage tree drawings were correlated with βIII-TUBULIN⁺ neurons produced (G). 80% of the divisions were asymmetric, giving rise to one neuron and one or more astrocytes and/or intermediate precursor cells (lineage trees J, K, and L), and 20% were symmetric, leading to the formation of two neurons (lineage trees H and I). The movie that accompanies this figure (Movie S1) concerns tracking of lineage tree (I). ?, cell lost from field of view. Scale bars, 40 μm. See also Figure S3 and Movies S1 and S2.

Figure 4

Formation of Three-Dimensional Spheres following Double CEND1 and NEUROG2 Forced Expression (Top) Protocol schematically representing all stages of astrosphere formation. (A–D) Snapshots of the following time points during 19 hr of live-cell imaging show the formation of GFP⁺ and dsRed⁺ clones: 0 hr (A), 9 hr (B), 12 hr (C), and 19 hr (D). (E–H) Lineage tree drawings indicate the creation of clones in early stages with the first division to happen between day 1 and 2 in all samples shown here: 21 hr (E), 14 hr (F), 19 hr (G), and 18 hr (H). (I) Three-dimensional colonies attached to the culture dish were present 48 hr after CEND1 and NEUROG2 double overexpression. (J–L) Live spheres isolated from the astrocytic culture and cultured in NSC medium (L) expressed both GFP (J) and dsRed (K) after 72 hr. (M and N) High NESTIN expression (M) and proliferative activity (N) in 10^th passage astrospheres. (O–Q) Upon growth factors removal, cells gave rise to βIII-TUBULIN⁺ neurons (O), O4⁺ oligodendrocytes (P), and GFAP⁺ astrocytes (Q). (R–U) Quantification and statistical analysis of immunostaining data indicated that the differentiation process toward neural progenitors (R), astrocytes (S), oligodendrocytes (T), and the proliferation capacity of astrospheres (U) was not statistically significant different from that of P5 subventricular zone-derived neurospheres. Nuclei were strained by TOPRO-3 (blue). Data were collected from at least three independent experiments. Scale bars, 40 μm. See also Movie S3.

Figure 5

MEF Reprogramming upon Forced Expression of CEND1 and/or NEUROG2 (Top) Schematic drawing of the protocol used for MEF reprogramming. (A, D, and G) 3 days after viral transduction, cells transduced with both GFP- and dsRed-crl viruses exhibited high FIBRONECTIN expression (A), which became diminished following CEND1 overexpression (D). (G) High transduction efficiency was observed in MEFs co-transduced with CEND1 and NEUROG2. (B, C, E, F, H, and I) In day 3 an alteration on the phenotype of the cell cultured can be noticed with CEND1- and NEUROG2-transduced cultures to have the biggest change (H), while CEND1⁺ cultures (E) have changed too compared to the control cultures (B). By day 14, cultures transduced with either CEND1 or CEND1 and NEUROG2 formed smaller (F) or bigger diameter spheres (I), depending on the molecule(s) being force expressed. No such cell types appeared in control cultures at the same time point (C). (J, L, and O) Cells expressing the NPC markers NESTIN and Sox2 in naive cultures (J) and following CEND1 (L) and CEND1 and NEUROG2 (O) transduction by the end of reprogramming period (day 14). (K, M, and P) A small percentage of βIII-TUBULIN⁺ neurons appeared in crl cultures by day 18 (K), while their number was significantly higher in CEND1-tranduced cultures (M) and in particular CEND1- and NEUROG2-transduced cultures (P). (N and Q) After 20 days, a subpopulation of βIII-TUBULIN⁺ cells was expressing the neuronal subtype-specific markers, GABA (N) and TH (Q) in CEND1 and CEND1- and NEUROG2-transduced cultures, respectively. Nuclei were strained by TOPRO-3 (blue). Scale bars, 40 μm.

Figure 6

MEF Culture Shift to Induced Neural Precursor Cell Culture and Neuronal Cells Shown via Immunohistochemical and Electrophysiological Analysis (A and B) Graphs indicating the percentages of NESTIN⁺ NPCs (A) and βIII-TUBULIN⁺ neurons (B) in the whole-cell population during reprogramming and neuronal differentiation stages both in naive cultures and upon CEND1 and/or NEUROG2 forced expression. (C and D) Percentages of GABA⁺ (C) and TH⁺ (D) neurons in the βIII-TUBULIN⁺ neurons population in naive cultures and upon CEND1 and NEUROG2 forced expression. All statistical data were collected from at least three independent experiments. (E and F) Representative whole-cell current response of CEND1- and/or NEUROG2-induced neurons. Step depolarizing pulses evoked fast activating and rapidly inactivating inward currents followed by outward non-inactivating ones. The inward component of the current response was abrogated by the selective sodium channel blocker TTX. (G) The outward component of the current response was blocked by the selective potassium channel blocker TEA. (H) Representative traces of whole-cell patch clamp recordings with CsCl in the recording pipette. When K+ ions were substituted by Cs+ ones in the patch pipette, depolarizing steps elicited only the fast inactivating inward current response but suppressed the outward ones. (I) The inward currents were also totally blocked by the presence of 1 μM of the selective voltage-gated sodium channel blocker TTX. (J) Representative recording of spontaneous current events of various amplitudes from a cell held at −60 mV. (K) The voltage protocol used to elicit whole-cell current responses. The holding potential was at −60 mV. The membrane was stepped at −120 before and after each depolarizing voltage step. (L) Whole-cell patch-clamp experiments were performed on neurons present in cell cultures for up to 42 days.

Figure 7

CEND1 and NEUROG2 Form a Positive Feedback Loop Inducing Neuronal Reprogramming (A and B) mRNA levels of Cend1 (A) and Neurog2 (B) 48 and 72 hr following their overexpression in astrocytic cultures as revealed by real-time qRT-PCR. (C) NEUROG2 overexpression resulted in elevated endogenous levels of CEND1. (D) CEND1 overexpression also induced endogenous NEUROG2 expression 48 hr following transduction. (E–K) Co-transduction of NEUROG2 together with a CEND1-silencing lentiviral vector expressing GFP (shCend1-GFP) (H–K) or the control lentivirus shLuc-GFP (E–G). In contrast with sh-Luc- and NEUROG2-transduced control cultures (E–G), where astrocytes expressing low levels of CEND1 (E, arrows) gave rise to GLAST⁺ radial glia (F, arrows) and βIII-TUBULIN⁺ neurons (G, arrows), the majority of sh-CEND1- and NEUROG2-transduced cells (H–K) retained their astrocytic morphology and GFAP expression (I and J, arrows) and did not express GLAST (H, arrows) or βIII-Tubulin (K, arrow). (J) Magnification of the squared cell in (I) exhibiting high GFAP expression. In the same cultures (H–K), single-transduced NEUROG2 cells trans-differentiated to GLAST⁺ radial glia (H, arrowhead) and βIII-TUBULIN⁺ neurons (K, arrowheads). GLT1, GLAST. Scale bars, 40 μm. (L) mRNA levels of four genes (Dl1, Ndp, POU4f1, Sox2) with differential mRNA levels in single CEND1- or NEUROG2-overexpressing astrocytic cultures 48 hr after transgene overexpression versus double-transduced or astrosphere cultures, as revealed by real-time qRT-PCR array analysis of 84 genes related to multipotency and neurogenesis. (M) The Brn2 mRNA levels of expression. (N and O) The mRNA levels of receptor Lrp-5 (N) and mRNA levels of LRP5’s downstream molecule β-catenin (O). The levels of Ndp, Lrp-5, and β-catenin are strongly upregulated in double-transduced and astrosphere cultures. By contrast, the mRNA levels of the neurogenic factor Brn2 are highly upregulated only in single-transduced cultures. (P) Schematic diagram of the canonical Wnt/β-catenin pathway, leading to subsequent activation of multipotency and/or proliferation genes. See also Figures S4 and S5. mRNA level quantification by qRT-PCR is based on three biological replicates.