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. 2023 May 1;31(3):264-275.
doi: 10.4062/biomolther.2022.140. Epub 2023 Jan 16.

Efficient Generation of Dopaminergic Neurons from Mouse Ventral Midbrain Astrocytes

Jin Yi Han  1 , Eun-Hye Lee  2   3 , Sang-Mi Kim  4   5 , Chang-Hwan Park  1   4   6
Affiliations

Efficient Generation of Dopaminergic Neurons from Mouse Ventral Midbrain Astrocytes

Jin Yi Han et al. Biomol Ther (Seoul). .

Abstract

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by tremors, bradykinesia, and rigidity. PD is caused by loss of dopaminergic (DA) neurons in the midbrain substantia nigra (SN) and therefore, replenishment of DA neurons via stem cell-based therapy is a potential treatment option. Astrocytes are the most abundant non-neuronal cells in the central nervous system and are promising candidates for reprogramming into neuronal cells because they share a common origin with neurons. The ability of neural progenitor cells (NPCs) to proliferate and differentiate may overcome the limitations of the reduced viability and function of transplanted cells after cell replacement therapy. Achaete-scute complex homolog-like 1 (Ascl1) is a wellknown neuronal-specific factor that induces various cell types such as human and mouse astrocytes and fibroblasts to differentiate into neurons. Nurr1 is involved in the differentiation and maintenance of DA neurons, and decreased Nurr1 expression is known to be a major risk factor for PD. Previous studies have shown that direct conversion of astrocytes into DA neurons and NPCs can be induced by overexpression of Ascl1 and Nurr1 and additional transcription factors genes such as superoxide dismutase 1 and SRY-box 2. Here, we demonstrate that astrocytes isolated from the ventral midbrain, the origin of SN DA neurons, can be effectively converted into DA neurons and NPCs with enhanced viability. In addition, when these NPCs are inducted to differentiate, they exhibit key characteristics of DA neurons. Thus, direct conversion of midbrain astrocytes is a possible cell therapy strategy to treat neurodegenerative diseases.

Keywords: Astrocytes; Dopaminergic neurons; Transdifferentiation; Ventral midbrain.

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Conflict of interest statement

CONFLICT OF INTEREST

There are no conflicts of interest.

Figures

Fig. 1
Fig. 1
NHB+A/5SA combinations are suitable for induction of DA neuronal conversion. (A) Schematic diagram outlining the iDAN conversion procedure. Mouse astrocytes were transduced with ASCL1, 5SA, NURR1-Lmx1A, and NHB. (B) Immunostaining of iDAN conversion on days 7,14, 21, and 28. Mouse astrocytes transduced with ALN, 5SALN, NHB+A, and NHB+5SA were converted into DA neurons and stained with TH (red) and TUJ1 (green). Over time, NHB+A/5SA produced more TH- and TUJ1-positive cells than ALN and 5SALN. (C) The numbers of TH- and TUJ1-positive cells/50 mm2 were counted on transduction days 7, 14, 21, and 28. Scale bar, 20 μm. *p<0.05. Error bars represent the SE.
Fig. 2
Fig. 2
iDAN conversion efficiency of VM-astrocytes is higher than that of CTX-astrocytes. (A) Mouse cortical (CTX) and ventral midbrain (VM) astrocytes 2 days after retroviral transduction (DPI 2). GFP-positive cells were observed in both CTX and VM astrocyte cultures. More GFP-positive cells were observed with VM astrocytes than CTX astrocytes. (B) The fold-change of GFP-positive cells was 3.8 times higher with VM astrocytes than CTX astrocytes. (C) Immunostaining of iDANs converted from CTX and VM astrocytes with NHB+ASCL1/5SA. After 7 and 14 days of conversion, cells were stained with TH (red) and TUJ1 (green). VM-iDANs had higher numbers of TH- and TUJ1-positive cells than CTX-iDANs. (D) TH- and TUJ1-positive cells were counted on transduction days 7 and 14. Scale bar, 20 μm. *p<0.05. Error bars represent SE.
Fig. 3
Fig. 3
SOD1 enhances iDANs conversion efficiency. (A) Immunostaining following NHB+A/5SA+SOD1 transduction on days 21, 28, 35, and 42. After 28 days of transduction, the fluorescence signal of TH- (red) and TUJ1- (green) -positive cells was the highest. (B) TH- and TUJ1-positive cells were counted on transduction days 21, 28, 35, and 42. (C) Proportion of TH- and TUJ1- double-positive cells versus TUJ1-positive cells at transduction day 28. Overexpression of NHB+A+SOD1 resulted in 88.4% TH-, and TUJ1-double-positive cells. Scale bar, 20 μm. Error bars represent the SE. *p<0.05.
Fig. 4
Fig. 4
SOX2 enhances iDANPC conversion efficiency. (A) A schematic outlining the timeline of the iDANPC conversion procedure. VM astrocytes were transduced with NHB, ASCL1, and EGFP or SOX2. Cells were passaged 7 days after transduction. (B) Immunostaining of iDANPCs converted from VM astrocytes with NHB, ASCL1 and EGFP or SOX2 and stained with NESTIN (red) and GFAP (green). Level of the NPC marker, NESTIN, was higher in the SOX2 co-overexpression group. (C) Proportion of NESTIN-positive cells (relative to DAPI staining). Overexpression of NHB, ASCL1, and SOX2 resulted in 8.4% NESTIN-positive cells. (D) Immunostaining of iDANPCs after subculture. Level of NESTIN (red) was increased and GFAP (green) decreased in iDANPCs according to passage numbers. (E) Number of NESTIN-positive cells in iDANPCs. Number of NESTIN-positive cells was highest in the P5 group overexpressing NHB, ASCL1, and SOX2. Scale bar, 20 μm. *p<0.05. Error bars represent SE.
Fig. 5
Fig. 5
VM-iDANs are mature and functional DA neurons. (A) Immunostaining of NHB+A/5SA+SOD1-induced DA neurons after 14 and 21 days of transduction, showing expression of midbrain specific markers (NURR1 and FOXA2), a synapse formation marker (SYNAPSIN 1), and neuronal markers (TUJ1, MAP2, and NEUN). (B) RT-PCR analysis of NHB+A/5SA+SOD1-induced DA neurons. On differentiation day 14, iDANs expressed the DA neuron marker. (C) Dopamine released from VM-iDANs 28 days after transduction. Supernatants were harvested after 48 h of cell incubation or 30 min of stimulation with 56 mM KCl. The highest release of dopamine in VM-iDANs was observed for NHB+A+SOD1-induced DA neurons. Scale bar, 20 μm. *p<0.05, **p<0.01. Error bars represent SE.
Fig. 6
Fig. 6
3D culture promotes iDANPC characteristics. (A) Schematic outlining the sequence of iDANPC conversion by 3D culture. Seven days after transduction, cells were passaged as 2D or 3D cultures, respectively. After 2D and 3D culture, cells were passaged on 24-well plates for staining. (B) Seven days after transfection, cells seeded in AggreWellTM400 plates settled and formed colonies. (C) Immunostaining of iDANPCs from AggreWellTM and single cells in expansion states. Expression of the NPC markers NESTIN (red), PAX6 (green), and SOX2 (green) is shown. AggreWellTM cell colonies expressed higher levels of the NPC markers than 2D cultures. (D) Statistical data of immunostaining in 3D culture compared to 2D culture. Scale bar, 20 μm. *p<0.05, **p<0.01. Error bars represent SE.
Fig. 7
Fig. 7
Differentiation of established iDANPCs into DA neurons. (A) Schematic outlining the sequence of iDANPC conversion and differentiation. Seven days after transduction, cells were passaged on AggreWellTM400 plates, and allowed to form colonies for 7 days. Colonies were seeded into 24-well plates for differentiation. (B) Immunostaining of the differentiated iDANPCs showing expression of the DA neuronal markers TH (red), FOXA2 (green), and NURR1 (green), and neuronal markers TUJ1 (green) and MAP2 (red). Scale bar, 20 μm.
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Grants and funding

ACKNOWLEDGMENTS This research was supported by a grant from the Individual Basic Science and Engineering Research Program (2019R1A2C2005681) of the National Research Foundation funded by the Ministry of Science and ICT in Korea.

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