Display Settings:

Items per page
We are sorry, but NCBI web applications do not support your browser and may not function properly. More information

Results: 6

1.
Figure 2

Figure 2. NGN2-induced neurons are functionally mature. From: Small Molecules Enable Neurogenin 2 to Efficiently Convert Human Fibroblasts to Cholinergic Neurons.

(a) Fibroblast-derived neurons fire repetitive action potentials by depolarizing current injections (n=10/10 recorded cells). (b-c) TTX-sensitive fast inward currents upon depolarization (n=7/7 recorded cells). (d-e) Sodium currents elicited at or above −30 mV, with a peak current at −1200 pA. (f-h) Inward currents induced by puffing inhibitory or excitatory neurotransmitters (n=3/4 for GABA, n=3/5 for glycine, and n=3/3 for kainate). (i) Induced neurons exhibit spontaneous activity indicating synaptic connections between neurons (n=19/19 of recorded cells).

Meng-Lu Liu, et al. Nat Commun. ;4:10.1038/ncomms3183.
2.
Figure 3

Figure 3. Neuronal conversion from human fibroblasts is accompanied by cell cycle exit. From: Small Molecules Enable Neurogenin 2 to Efficiently Convert Human Fibroblasts to Cholinergic Neurons.

(a) Experimental design for data in (b-c). Cells were incubated with BrdU for 2 hr before immunocytochemistry. FM, fibroblast medium; NM, neuronal induction medium containing FSK and DM. (b-c) Treatment with neuronal induction medium quickly stopped proliferation of cells transduced with either GFP or NGN2-IRES-GFP viruses (means ± s.e.m., n=15 randomly selected 20x fields from triplicate samples, *p < 0.001 by Student’s t-test). Scale, 50 μm. (d) Experimental design for data in (e-g). Cells were treated with BrdU for the indicated periods of time. (e-g) A majority of the cells were labeled with BrdU if treated prior to neuronal conversion (BrdU/FM-NM), whereas cells were not labeled by BrdU if treated one day after switching to neuronal induction medium (NM/BrdU). Quantitative data are means ± s.e.m. from 20 randomly selected 20x fields from triplicate samples. n.d., not detected. Scale, 50 μm.

Meng-Lu Liu, et al. Nat Commun. ;4:10.1038/ncomms3183.
3.
Figure 6

Figure 6. Efficient generation of cholinergic neurons from postnatal and adult human skin fibroblasts. From: Small Molecules Enable Neurogenin 2 to Efficiently Convert Human Fibroblasts to Cholinergic Neurons.

(a) SOX11 and FGF2 facilitate NGN2 to convert postnatal (GM05565 and GM03813) or adult (GM03814, GM07522, and ND29563) human skin fibroblasts to neurons. Tuj1+ cells were quantified from triplicate samples at 21 dpi (means ± s.e.m.). (b-c) Conversion efficiency normalized to total number of seeded cells (b) or NGN2-expressing cells (c). The total number of Tuj1+ cells from triplicate samples was quantified at 21 dpi (means ± s.e.m.). A longer treatment of DM/FGF2 enhances conversion efficiency. (d) Representative images of converted Tuj1+ neurons from postnatal (GM03813) and adult (GM03814, the mother of GM03813) fibroblasts at 21 dpi. (e-f) Quantification of converted neurons expressing ChAT (e, 81.9%-90.7%) or HB9 (f, 78.0%-85.9%). The total number of Tuj1+, ChAT+, or HB9+ cells from triplicate samples was quantified at 21 dpi (means ± s.e.m.). (g-j) Induced neurons from skin fibroblasts (GM07522) express pan-neuronal marker MAP2 (g) and presynaptic marker SYT1 (h). (i-j) Converted neurons from skin fibroblasts (GM03813) are cholinergic (ChAT+, i) and express markers for early motor neurons (HB9+, j). Arrows show cells stained negative for the indicated neuronal markers. Scales, 50 μm.

Meng-Lu Liu, et al. Nat Commun. ;4:10.1038/ncomms3183.
4.
Figure 5

Figure 5. Cholinergic motor neuron-like cells converted from human fetal fibroblasts. From: Small Molecules Enable Neurogenin 2 to Efficiently Convert Human Fibroblasts to Cholinergic Neurons.

(a-d) Immunocytochemistry analysis of induced neurons with markers for cholinergic motor neurons, including HB9 (91.7%, n=325), ISL1/2 (90.2%, n=347), ChAT (98.5%, n=325), and VAChT (98.2%, n=273). GFP expression indicates NGN2-transduced cells. Control virus-transduced fibroblasts were stained negative for each of these markers. Arrows indicate cells stained negative for HB9 or ChAT. (e-i) Gene expression analysis by qRT-PCR during the reprogramming process (means ± s.e.m., n=3 at each time point). It was normalized to that of house-keeping gene Hprt. Samples from normal adult human brain (Brn) and spinal cord (SC) were used as controls (n=1). **p < 0.01 by Student’s t-test. (j) Immunocytochemistry analysis of synaptic connections between hiCNs and myotubes in co-culture. A multinucleated myotube is outlined. α-BTX, α-bungarotoxin; SYT1, synaptotagmin 1. (k) Quantification of rhythmic contractions of myotubes after co-culturing with hiCNs. Such contractions can be blocked by curare, a competitive inhibitor of acetylcholine receptor (n=5 independent myotubes). Scales, 20 μm for (a-b), 50 μm for (c-d), and 10 μm for (j).

Meng-Lu Liu, et al. Nat Commun. ;4:10.1038/ncomms3183.
5.
Figure 1

Figure 1. NGN2 induces rapid and efficient neuronal conversion of human fetal fibroblasts. From: Small Molecules Enable Neurogenin 2 to Efficiently Convert Human Fibroblasts to Cholinergic Neurons.

(a) FSK but not any other tested small molecules synergizes with NGN2 to generate Tuj1+ cells from IMR-90 fetal fibroblasts. GFP indicates cells transduced with NGN2-expressing virus. FSK, forskolin; RA, retinoic acid; VPA, valproic acid; NaB, sodium n-butyric acid; TSA, trichostatin A; SB, SB431542; DM, dorsomorphin. (b) Quantification of Tuj1+ cells 5 days post chemical treatments (means ± s.e.m., n=40 randomly selected 20x fields from triplicate samples). (c) DM promotes conversion efficiency, neuronal survival and maturation. Cells were counted 10 days post treatment (means ± s.e.m., n=30 random 20x fields from triplicate samples). (d) Time-dependent maturation of converted neurons by NGN2/FSK/DM (means ± s.e.m., n=10 random 20x fields from triplicate samples). (e-f) Representative morphology of induced neurons at 7 or 30 dpi. (g-l) Converted cells express markers for mature neurons. (m-n) Induced neurons express glutamatergic (vGlut1) and presynaptic (synaptotagmin 1, SYT1) markers, which showed a punctate staining pattern. (o) GABA is rarely detected in converted neurons. Scales, 50 μm for (a, e-k, n-o), 20 μm for (l, m).

Meng-Lu Liu, et al. Nat Commun. ;4:10.1038/ncomms3183.
6.
Figure 4

Figure 4. Neuronal conversion from human fibroblasts does not require long-term expression of NGN2. From: Small Molecules Enable Neurogenin 2 to Efficiently Convert Human Fibroblasts to Cholinergic Neurons.

(a) qRT-PCR analysis of retrovirus-expressed NGN2 (vNGN2) and the induction of endogenous NGN2 (endoNGN2) at the indicated time points after retroviral transduction of human fetal fibroblasts (means ± s.e.m., n=3 at each time point). Samples from normal adult human brain (Brn) and spinal cord (SC) were used as controls (n=1). (b) Experimental design using doxycycline (Dox) inducible expression system. Cells (5,000/well in 48-well plate) were cotransduced with lentivirus constitutively expressing rtTA3G and Dox-inducible lentivirus(iLV) expressing NGN2-IRES-GFP under the TRE3G promoter. One day post transduction, Dox (0.1 μg/ml) was applied for the indicated periods of time and then withdrawn. The transduction efficiency was about 8% indicated by initial GFP expression. Tuj1-expression was analyzed 35 days later. (c) qRT-PCR analysis of exogenous NGN2 expression, which returns to basal level 6 days after Dox withdrawal (n=1). (d) NGN2 expression is no longer required after neuronal conversion at 21 dpi, although it increases total number of converted neurons at earlier time points. Tuj1+ cells were quantified from a whole well from triplicate samples (means ± s.e.m., n=3). *p=0.04, **p=0.003, and ***p=0.0003 by Student’s t-test.

Meng-Lu Liu, et al. Nat Commun. ;4:10.1038/ncomms3183.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk