Table 2.
Representative In Vitro and In Vivo Direct AtN Conversion and Reprogramming Studies.
| Induction Factor(s) | Vector/ Delivery System |
Cell Type/ Anatomical Target |
Induction Efficiency (%) | Criteria | iN Phenotype/ Criteria |
iN Features | |
|---|---|---|---|---|---|---|---|
| In vitro astrocyte to neuron reprogramming | |||||||
| NeAL218 + MP * [26] | Lentivirus carrying rtTA [26] | ATCC (SVGp12, cat. n CRL86-21), midbrain (hIAs) | 30.97 ± 5.3 | TH+, MAP2+ (84.6 ± 1.9%), TUBB3+ |
Dopaminergic (100% of iN)—DDC, SLC6A3, FOXA2, EN1, and SLC18A | Simple neuron-like morphologies and lack emDAs membrane properties | |
| NeAL218 + RTMP * [26] | ATCC (SVGp12, cat. n CRL86-21), midbrain (hIAs) | 16.48 ± 8.6 | TH+, TUBB3+, MAP2+, SYN+ | Dopaminergic (100% of iN)—DDC, SLC6A3, ALDH1A1, and KCNJ6 | Ca2+ response upon depolarization (55 mM KCl), generate AP, sEA + AP at 13–17 days, current clamp recordings show different firing properties upon current injection (none, single AP, multiple AP), and 2/7 (≈29%) generate multiple AP | ||
| Lonza (normal human astrocytes, cat. n CC-2565), hPAs | 12.4 ± 2.7 | TH+, MAP2+, RBFOX3+ | Dopaminergic (100% of iN)—DDC, SLC6A3, ALDH1A1, KCNJ6, and PBX1 | ||||
| In vitro astrocyte to neuron conversion | |||||||
| Ascl1 (Mash1) [29,88] |
Lentivirus FUGW [29] | Isolated from P5–P7 mice, postnatal dorsal midbrain | 76.8 ± 6.4 | Tuj1+ MAP2+, and Synapsin I+ |
Glutamatergic (19.4%)—blocked by CNQX, GABAergic (8/38, ≈21%)—blocked by Bicuculline |
Produce AP and sPSC in 85.3% | |
| Retroviral VSV-G [88] | C57BL/6 mice P5–P7, pNCC |
37 ± 11% and 14 ± 2% Tuj1+, and >40% TuJ1- |
TuJ1+ | No TuJ1+/Tbr1+, no clear nuclear staining for Ascl1 (Mash1) |
Intrinsic excitability, generate typical neuronal AP, and virtual absence of spontaneous synaptic input | ||
| Neurog2 (Ngn2) [88,89] | Retroviral VSV-G [88] | C57BL/6 mice P5–P7, pNCC |
>85%, 71 ± 16%, and 16 ± 18% clones TuJ1+, and ~10% clones TuJ1- |
TuJ1+ | Glutamatergic (≈33%)—TuJ1+/Tbr1+, blocked by CNQX GABA (polysynaptic, UD)— >5 ms delay, blocked by both CNQX and Bicuculline |
Fire repetitive AP, ↑ negative resting mV, ↓ IR, ↑ AP amp over time, functional but ↓ PS response, and not generate SR from neighboring neurons | |
| pCAG-IRES-DsRed (self- silencing, long-acting) [89] |
C57BL/6J or GLAST::CreERT2/Z/EG mice P5–P7, pNCC |
70.2 ± 6.3%, | BIII tubulin+, GFAP- | Glutamatergic (58.3%)—BIII tubulin+/vGlut1+ puncta (85.4 ± 5.0%) GABA (0%) |
MAP2 in 2–3 weeks and Ca2+ transients (63.8%) | ||
| In neurosphere [89] | 91.4 ± 2.2% | MAP2+ | Glutamatergic—MAP2+/vGlut1+, AC/SC (9/21, ≈43%), CNQX-sensitive sSC (8/30, ≈27%) | Low IR | |||
| Dlx2 [89] | pCAG-IRES-DsRed (self- silencing, long-acting) [89] |
C57BL/6J or GLAST::CreERT2/Z/EG mice P5–P7, pNCC |
35.9 ± 13.0% | BIII tubulin+, MAP2+ | GABAergic—Autapses, vGlut1-, BIII tubulin/vGaT+ (33.7 ± 3.6%), sSC with slow decay time (9/33, ≈27%), AR blocked by Bicuculline | Neuron morphology, fire AP, distinct firing patterns (regular, stuttering, and low-threshold), 7/9 (≈78%) immature firing pattern, and 2/9 (≈22%) mature interneuron-like firing pattern | |
| In neurosphere [89] | 94.7 ± 0.3% | MAP2+ | GABAergic—MAP2+/vGaT+ puncta, slow decay time (9/10, ≈90%, UD) | ↓ IR and no Ca2+
transients |
|||
| Induction Factor(s) |
Vector/
Delivery System |
Animal Model/Sex |
Anatomical
Target |
Direct Reprogramming
Efficiency (%) |
Criteria |
iN Phenotype/
Criteria |
iN Features |
| In vivo astrocyte to neuron reprogramming | |||||||
| ALN * [90] | Cre-inducible AAV5/injection | Adult GFAP-Cre mice (P84–P112) | Striatum | 46.8 ± 2.9 | NeuN+ | Glutamatergic—vGlut1+ (16%) GABAergic—GAD65/67+ (68%) |
rMP (−61.4 ± 9.7 mV), AP mean amp (33.5 ± 2.29 mV), and AP threshold (25 ± 7.19 pA) |
| NeAl218 * [26] | Tet-regulated NeAL218 lentiviruses/stereotactic needle injection | Adult Tg(GFAP-tTA)110Pop/J mice (P60–P180) | Ipsilateral striatum | 14.63 ± 8.5 | TH+ | Dopaminergic—TH+/SLC6A3+, RBFOX3+, NR4A2+, and PBX1+ | TH+/SLC6A3+ iNs produced Ih |
| In vivo astrocyte to neuron conversion | |||||||
| Ascl1 [29] |
AAV/ micropipette injection [29] |
Adolescent WT mice (P12–P15), M + F | Dorsal midbrain | 93.1 ± 1.7 | NeuN+ | GABAergic—NeuN+/Gad1+ (13.2 ± 4.2%) Glutamatergic—NeuN+/VGLUT2+ (6.5 ± 2.2%) |
Producing AP, sPSC observed, IOC in VCM, MΩ (177.3 ± 16.6), and ↓ RMP (−61.9 ± 1.0) |
| Adult WT mice (P60), M + F |
92.1 ± 1.5 | NeuN+ | GABAergic—NeuN+/Gad1+ (11.7 ± 4.0%) Glutamatergic—NeuN+/VGLUT2+ (6.3 ± 1.3%) |
Producing AP, sPSC observed, IOC in VCM, MΩ (240.0 ± 81.9), and ↓ RMP (−61.0 ± 1.2) | |||
| Striatum | 64.4 ± 3.4 | NeuN+ | GABAergic (according to electrophysiological test performed) | Fire APs in CCM (13/16, ≈81%), sEPSC and sIPSCs (12/16, ≈75%), and IOC in VCM (15/16, ≈94%) | |||
| Somatosensory cortex | 93.9 ± 1.2 | NeuN+ | Glutamatergic or GABAergic (according to electrophysiological verification) |
Record 163.3 ± 35.9 MΩ, dMP (−67± 2.2 mV), APs, IOC, sEPSC, and sIPSCs | |||
| AAV-FLEX/ micropipettes injection [29] |
Adult Aldh1l1–Cre transgenic mice (P60), M + F | Dorsal midbrain | 90.1 ± 2.1 | NeuN+ | GABAergic (according to electrophysiological verification) |
Exhibit firing patterns identical to midbrain endogenous GABAergic neurons | |
| AAV/needle injection [29] |
Injured dorsal midbrain | 54.2 ± 6.9 | NeuN+ | Glutamatergic or GABAergic (according to electrophysiological verification) |
424.7 ± 88.7 MΩ, rMP (−61.2 ± 1.6 mV), IOC in VCM, rAPs fired in CCM, sEPSC, and sIPSCs | ||
| NeuroD1 [21,22,23] | AAV/stereotactic needle injection and infusion pump [21] | Adult Macaca mulatta (9–21 years old), M | Cortex | 94.4 ± 5.5 | NeuN+/ Tbr1+ |
Glutamatergic—Tbr1+, projection neurons | ↑ SV2 and significantly recovered MAP2 |
| AAV9/ stereotactic needle injection [23] |
Adult WT mice (P90–P180), M + F |
Cortex | 90.6 ± 5.2 | NeuN+ | Glutamatergic—vGlutT1+ GABAergic—GAD67+ |
↑ SMI32, ↑ vGluT1 and GAD67, large Na+/K+ currents (13/15, ≈87%), rAPs (7/10, ≈70%), glutamatergic SE (10/13, ≈77%), and GABAergic SE (9/13, ≈69%) | |
| Cre-FLEX AAV/needle injection [22] |
Adult WT mice (P60–P120), M + F | Stab-injured dorsal horn T10 | ~95.0 | NeuN+ | Glutamatergic—NeuN+/Tlx3+ (62.6 ± 3.3%) GABAergic—NeuN+/Pax2+ (8.8 ± 1.3%) |
rAPs, large Na+/K+ current, robust spontaneous EPSCs, and no difference in Na+ current and sEPSCs compared with neighboring native neurons | |
| Contusive SCI model T10 acute phase | ~55.0 | NeuN+ | Glutamatergic—Neu+/Tlx3+ in dorsal horn | ↑ SV2 | |||
| Contusive SCI model T11–T12 chronic phase | >95.0 | NeuN+ | Glutamatergic—Neu+/Tlx3+ in dorsal horn | ↑ SV2 | |||
| Neurog2 [24] | AAV/stereotactic needle injection | Adult WT mice | Dorsal midbrain | 96.3 ± 1.7 | NeuN+ | Glutamatergic—NeuN+/VGLUT2+ (64. 97 ± 8.04%) GABAergic—NeuN+/Gad1+ (2.26 ± 2.07%) |
Multiple APs, IOC in VCM, EPSC, MC and the IR of iN are largely comparable with local neurons, and neuronal profile |
| Dorsal horn T8–T10 | 80.11 ± 5.42 | NeuN+ | Glutamatergic—Tlx3+ (50.9 ± 8.8%) GABAergic—Pax2+ (38.5 ± 8.3%) |
Produce IOC in VCM, multiple APs (9/11, ≈82%; ↓ AP amp), and MC and iR comparable to native neurons | |||
| AAV/injection from L1–L2 dorsal surface |
Transected SC T8–T10 | 41.62 ± 22.82 | NeuN+ | Data not provided | Data not provided | ||
| Ptbp1 knockout [35] | AAV-GFAP-CasRx-Ptbp1 with gRNAs 5 + 6 for Ptbp/stereotactic injection | Adult C57BL/6 mice (~P70) | Striatum | 48.00 ± 10.00 | NeuN+ | Glutamatergic—50% iNs glutaminase+ | Data not provided |
| Ipsilateral striatum/PD model | 32.00 ± 7.00 | TH+ | Dopaminergic—TH+/DAT+ (31 ± 7%), ~15% TH+/DDC+, ~37% TH+/FOXA2+ iNs were ALDH1A1+, GIRK2+, and CB– |
rAPs (20/22, ≈91%) in response to depolarizing current injection in the CCM, sPSC observed in VCM (Vc = −70 mV), delayed voltage rectification induced by Ih (4/10, 40%), and majority iNs were VMAT2+ | |||
| NeuroD1 + Dlx2 [22,27] | rAAV2/5/stereotactic bilateral needle injection [27] |
Adult WT mice (P60–P140), M + F | Striatum | 72.7 | NeuN+ | MSN—NeuN+/DARPP32+ (55.7%) GABAergic—NeuN+/GAD67+ (83.9%) GABAergic—NeuN+/GABA+ (85.0%) Interneurons—NeuN+/PV+ (9.6%) NeuN+/SST+ or NPY+ or CalR+ (<5%) |
Data not provided |
| Adult R6/2 transgenic mice (P60–P150), M + F | 78.6 | NeuN+ | MSN, GABAergic, and interneuron; additional expression: DARPP32 (56.6%), GAD67 (82.4%), GABA (88.7%), PV (8.4%), and <5% (SST, NPY, CalR) | iNs rAPs (17/18, ≈94%), 72.2% firing at <80 Hz, 22.2% firing at >80 Hz, detected sEPSCs and sIPSCs in all iN, and ↑ iR, ↓ cC, ↓ RMP, and ↓ AP amp compared with control | |||
| Middle-aged YAC128 transgenic mice (15 months), M + F | 50.0 | NeuN+ | MSN, GABAergic, and interneuron; additional expression: DARPP32 (29.8%), GABA (half), and PV (3.9%) | Data not provided | |||
| Cre-FLEX AAV/needle injection [22] |
Adult WT mice (P60–P120), M + F | Stab-injured dorsal horn T11–T12 | N/A | Tlx3+ Pax2+ |
Glutamatergic—Tlx3+ (56.2 ± 3.4%) GABAergic—Pax2+ (32.5 ± 2.1%) |
Data not provided | |
| Ascl1 + Nurr1 [41] |
FLEX-switch AAV/ microinjection |
Adult mGFAP-Cre mice (P60–90), M + F | Injury cortex model | 40.0 (24 dpi) 70.0 (72 dpi) |
NeuN+ | iNs variable morphology | * Ascl alone served as a control and was shown to have a conversation efficiency of ≈20.0% (NeuN+) |
| Neurog2 + Nurr1 [41] | 53.0 (24 dpi) 80.0 (72 dpi) |
NeuN+ | NeuN+/CUX1+ iNs in upper layer, NeuN+/CUX+ iNs in deeper layer; both displayed stereotypical pyramidal-shaped cell soma; single and combinatorial labeling for CUX1, SATB2, and BRN2+ iNs in upper layers FOXP2+, CTIP2+, TLE4+, and TBR1+ iNs in lower layer |
rMP, iR, APs comparable to endogenous neurons, and E/I input blocked by NBQX. * Nurr1 alone served as a control and was shown to have a conversation efficiency of ≈20.0% (NeuN+) |
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* ALN—Ascl1, Lmx1a, and Nurr1; * NeAl218—NeuroD1, Ascl1, Lmx1a, and miR218 (for abbreviations and iN biomarker information, see Supplementary Materials, Tables S1 and S2). Notes: (1) In vitro astrocyte to neuron (AtN) direct reprogramming using a cocktail of induction factors and specific molecular protocols to produce “induced neurons” (iN). These protocols were reported to unwind DNA so that transcription factors could enter and induce changes in phenotype. (2) In vitro AtN direct conversion was performed by using up to two transcription factors to generate iNs. (3) In vivo AtN direct reprogramming was performed by using ≥3 transcription factors or small molecules affecting multiple epigenetic and genetic elements to reprogram astrocytes into reported glutamatergic, GABAergic, or dopaminergic iNs. (4) In vivo AtN direct conversion used up to two transcription factors to generate reported glutamatergic, GABAergic, or dopaminergic iNs. (5) The delivery system of induction factors, animal sex (M: male; F: female), and anatomical target for each study are exhibited. (6) Reported reprogramming/conversion efficiency (%) is presented based on the specific criteria of each study for defining an iN. (7) Essential information on the functional assessment of iNs is included.