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Method Article
Here, we describe a protocol for engineering chemically reprogrammed stem cells to achieve precise neuronal modulation by differentiating these cells into dopaminergic precursor cells, transplanting them into mouse models of Parkinson's disease, and evaluating behavioral and electrophysiological outcomes to confirm the successful integration and functional effectiveness of the transplanted cells.
The integration of designer receptors exclusively activated by designer drugs (DREADD) with stem cell-based therapies presents an advanced strategy for precise neuronal modulation. Here, we utilized CRISPR-engineered human reprogrammed stem cells expressing excitatory (hM3Dq) or inhibitory (hM4Di) DREADD receptors to evaluate the functional integration and modulation of transplanted dopaminergic precursors in a murine model of Parkinson's disease (PD). Key steps included generating non-fusion DREADD-expressing stem cell lines, differentiating them into midbrain dopaminergic precursors, and transplanting these cells into the striatum of 6-hydroxydopamine (6-OHDA)-lesioned mice. We conducted behavioral assessments and electrophysiological recordings to analyze the effects of the transplanted cells. Behavioral tests, such as the cylinder test, demonstrated significant modulation of motor function following clozapine-N-oxide (CNO) administration. Specifically, activation of hM4Di reduced contralateral forelimb movement, whereas activation of hM3Dq was associated with enhanced motor behavior. Electrophysiological recordings revealed distinct synaptic responses. hM4Di activation increased interevent intervals and decreased peak amplitudes of spontaneous excitatory postsynaptic currents (sEPSCs), whereas hM3Dq activation decreased interevent intervals and increased peak amplitudes, reflecting enhanced excitatory signaling. In summary, the integration of behavioral and electrophysiological assessments validates the precise functional incorporation of engineered chemically reprogrammed stem cells into host neural circuits.
Designer receptors exclusively activated by designer drugs (DREADD) are engineered G-protein coupled receptors that can be selectively activated by otherwise inert synthetic ligands1. The chemogenetic approach has become an essential tool in neuroscience by enabling researchers to investigate neural circuit connectivity with high precision and enhancing our understanding of cellular functions both in vivo and in vitro through the selective activation or inhibition of specific brain regions or cell types2,3.
Stem cell-based therapy presents a promising strategy for treating neurodegenerative diseases. The efficacy of graft cells relies on proper integration, survival, and functional contribution to host tissues. Uncontrolled cellular activity can lead to negative consequences, including tumorigenesis4; necessitating precise control of these cells post transplantation. Leveraging DREADD technology in human reprogrammed stem cells and derived neurons provides a means to precisely control neuronal activity via the administration of the designer drug CNO2,5. In the context of Parkinson's disease (PD), which is characterized by the loss of dopaminergic neurons, manipulating the activity of stem cell-derived dopaminergic neurons is crucial for investigating their synaptic inputs and projection patterns in rodent models6,7,8,9,10. Incorporating excitatory hM3Dq and inhibitory hM4Di receptors into these models enables precise modulation of neuronal activity11,12.
The combination of animal behavioral assessments and electrophysiological recordings allows for a comprehensive evaluation of the effects of chemogenetic modulation on transplanted cells in vivo13. Behavioral assessments, including apomorphine-induced rotation, the cylinder test, and the rotarod test, evaluate motor coordination and provide insights into changes in motor function associated with experimental models of PD14. Electrophysiological techniques, such as patch-clamp recordings, enable real-time monitoring of synaptic responses and action potentials, providing a comprehensive view of how transplanted cells integrate into existing neural networks15. By combining behavioral assessments with electrophysiological evaluations, we can investigate how chemogenetic modulation affects the integration and functionality of these cells within host neural circuits16. Preliminary findings suggest that CNO administration effectively modulates neuronal activity in transplanted cells, resulting in improved functional outcomes in animal models.
In this protocol, human reprogrammed stem cells were engineered to express hM3Dq or hM4Di receptors by using clustered regularly interspaced short palindromic repeats (CRISPR) technology. After differentiating modified reprogrammed stem cells into midbrain dopaminergic precursor cells, these cells were transplanted into mouse models of PD to assess their integration and functional regulation within the host neural circuits using behavioral assessments and electrophysiological recordings.
All animal experiments were performed in accordance with the guidelines set forth by the Beijing Association for Laboratory Animal Science and the National Institutes of Health for the Care and Use of Laboratory Animals. Human peripheral blood mononuclear cells (PBMCs) were obtained from a healthy donor with written informed consent as described in a previous study17.
1. Construction of non-fusion DREADD stem-based cell lines
2. Transplanted DREADD stem cell-derived precursor cells into PD model mice
3. In vivo electrophysiological profiling of chemogenetically modulated cells
Figure 1 shows the key steps of this methodological approach for engineered human reprogrammed stem cells expressing excitatory (hM3Dq) or inhibitory (hM4Di) DREADD receptors to evaluate the functional integration and modulation of derived dopaminergic precursors in a mouse model of Parkinson's disease (PD). Figure 2 outlines the CRISPR/Cas9-mediated gene knock-in strategy for introducing non-fusion constructs of hM4Di-T2A-ZsGreen and hM3Dq-T2A-ZsGreen into ...
This protocol utilized CRISPR technology to engineer human reprogrammed stem cells to express excitatory hM3Dq and inhibitory hM4Di receptors. The modulation of neuronal activity by CNO was assessed through cell transplantation into mouse models of Parkinson's disease, accompanied by behavioral assessments and electrophysiological recordings.
The first critical step in generating stably expressed, non-fusion DREADD constructs in chemogenetically reprogrammed stem cells involves the T2A-ZsGreen...
The authors have no conflicts of interest to disclose.
This work was supported by the Beijing Natural Science Foundation (7242068), National Natural Science Foundation of China (82171250), Beijing Municipal Health Commission Fund (PXM2020_026283_000005) and The Project for Technology Development of Beijing-affiliated Medical Research Institutes (11000023T000002036310).
Name | Company | Catalog Number | Comments |
2 x Rapid Taq Master Mix | Vazyme | P222-01 | used for genotyping analysis |
2×Seamless Cloning Mix | Biomed Gene Tech. | CL117-01 | used for plasmid construction |
6-OHDA | Sigma-Aldrich | H4381 | used for establishing PD mice model |
AAVS1-Pur-CAG-EGFP | Addgene | 80945 | used as control |
AAVS1-Pur-CAG-hM4Di-mCherry | Addgene | 80947 | original plasmid for construction of hM4Di-T2A-ZsGreen |
AAVS1-Pur-CAG-hM3Dq-mCherry | Addgene | 80948 | original plasmid for construction of hM3Dq-T2A-ZsGreen |
AAVS1-CAG-hM4Di-T2A-ZsGreen | N/A | N/A | Constructed donor plasmid based on #80947 |
AAVS1-CAG-hM3Dq-T2A-ZsGreen | N/A | N/A | Constructed donor plasmid based on #80948 |
Accutase | Invitrogen | A11105-01 | Used for digesting cells |
AMAXA Nucleofector | Lonza | AAD-1001S | |
Apomorphine | Sigma-Aldrich | A4393 | |
Artificial cerebrospinal fluid (ACSF) | N/A | N/A | 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1.25 mM NaH2PO4, 1 mM MgSO4, 25 mM glucose, and 26 mM NaHCO3 |
Ascorbic acid | Sigma-Aldrich | 1043003 | |
B-27 Supplement | Gibco | 17504044 | |
BDNF | Peprotech | 450-02 | |
cAMP | Sigma-Aldrich | D0627 | |
CHIR99021 | Yeasen | 53003ES10 | |
Clozapine-N-oxide | Enzo | BML-NS105 | |
DAPT | Sigma-Aldrich | D5942 | |
Desipramine | Sigma-Aldrich | D3900 | |
D-glucose | Sigma-Aldrich | G5767 | |
DMEM/F12 | Gibco | 11330-032 | |
DMEM/F12 | Gibco | 11320-033 | |
DMSO | Sigma-Aldrich | D2650 | |
FGF8 | Peprotech | 100-25 | |
GDNF | Peprotech | 450-10 | |
GlutaMax | Gibco | 35050-061 | |
Hank’s Balanced Salt Solution (HBSS) | Gibco | 14175095 | |
Human leukemia inhibitory factor (hrLIF) | Millipore | LIF1010 | |
Iced intracellular fluid | N/A | N/A | 130 mM K-gluconate, 16 mM KCl, 0.2 mM EGTA, 2 mM MgCl2, 10 mM HEPES, 4 mM Na2-ATP, 0.4 mM Na3-GTP, 0.3% of neurobiotin |
KnockOut Serum Replacement | Gibco | 10828028 | |
Laminin | Roche | 11243217001 | |
Micropipette Puller | Sutter Instrument Company | P-1000 | |
N-2 Supplement | Thermo Fisher | 17502048 | |
Neurobasal-A Medium | Gibco | 10888-022 | |
Non-essential amino acids (NEAA) | Gibco | 11140-050 | |
PBS | Gibco | 10010023 | |
pCLAMP 11 software suite | Molecular Devices | N/A | Patch-clamp electrophysiology data acquisition and analysis software |
Phase 1 differentiation medium | N/A | N/A | 96% DMEM/F12 (Gibco, 11320-033), 1% B-27 Supplement, 1% N-2 Supplement, 1% NEAA, 1% GlutaMax, 1 µM SAG1, and 100 ng/mL FGF8 |
Phase 2 differentiation medium | N/A | N/A | 96% DMEM/F12 (Gibco, 11320-033), 1% B-27 Supplement, 1% N-2 Supplement, 1% NEAA, and 1% GlutaMax, 10 ng/mL BDNF, 10 ng/mL GDNF, 1 ng/mL TGF-βIII, 10 µM DAPT, 0.2 mM ascorbic acid, and 0.5 mM cAMP. |
Poly-D-lysine hydrobromide (PDL) | Sigma-Aldrich | P7886 | |
Primers for genotyping | N/A | N/A | Insertion Foward: TCTTCACTCGCTGGGTTCCCTT; Insertion Reverse: CCTGTGGGAGGAAGAGAAGAGGT; Homozygosity Foward:CGTCTCCCTGGCTTTAGCCA; Homozygosity Reverse: GATCCTCTCTGGCTCCATCG |
pX458 | Addgene | 152199 | |
SAG1 | Enzo | ALX-270-426-M01 | |
SB431542 | Yeasen | 53004ES50 | |
sucrose-based artificial cerebrospinal fluid (s-ACSF) | 234 mM sucrose, 2.5 mM KCl, 26 mM NaHCO3, 1.25 mM NaH2PO4, 11 mM Dglucose, 0.5 mM CaCl2, and 10 mM MgSO4 | ||
Stem cell culture media | N/A | N/A | 48% DMEM/F12 (Gibco, 11330-032) and 48% Neurobasal, with the addition of 1% B27, 1% N2, 1% NEAA, 1% GlutaMax, 10 ng/mL hrLIF, 2 µM SB431542, and 3 µM CHIR99021 |
TGF-βIII | Peprotech | 100-36E | |
Transplantation buffer | N/A | N/A | HBSS buffer with 5 g/L D-glucose, 100 ng/mL BDNF, 100 ng/mL GDNF, and 0.2 mM ascorbic Acid |
Vibratome | Leica | VT1000 S | |
Whole-cell patch-clamp | Molecular Devices | MultiClamp700B |
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