Name: Video: Transplantation of Human Stem Cell-Derived GABAergic Neurons into the Early Postnatal Mouse Hippocampus to Mitigate Neurodevelopmental Disorders
Uploaded: 2022-11-11T13:40:00.000+00:00
Duration: 5 min

This project was funded by the Swedish Research Council (Grant Number: 2016-02605, M.A.), the Swedish Brain Foundation F02021-0369 (M.A.), the Crafoord Foundation (M.A.), and the European Union Horizon 2020 Programme (H2020-MSCA-ITN-2016) under the Marie Sk&#322;odowska-Curie Innovative Training Network project Training4CRM No. 722779 (M.K.). We are extremely grateful for the help from Andr&#233;s Miguez, from Josep Maria Canals&#39; lab (Laboratory of Stem Cells and Regenerative Medicine, University of Barcelona), for teaching stereotaxic cell transplantation in P2 newborn mice, and Mackenzie Howard, group leader at the University of Texas at Austin, for the advice and preliminary coordinates for cell transplantation into the hippocampus of P2 newborn mice. We thank Susanne Geres for assisting with animal care and Ling Cao for the help with processing tissue, as well as students that have contributed in one way or another to the study and specifically&#160;Diana Hatamian. Finally, some of the graphics used to illustrate this paper were created with BioRender.com.

All experimental procedures were approved by the Malm&#246;/Lund Animal Research Ethics Board, ethical permit number 12548-19, and conducted in agreement with the Swedish Animal Welfare Agency regulations and the EU Directive 2010/63/EU for animal experiments. C57BL6/J and Contactin-associated protein-like 2 (Cntnap2)&#160;knock-out (KO) mice, both males and females, were used at postnatal day (P) 2 for the present study. Human embryonic stem cells (hESCs) were used. The animals and stem cells were obtained from commercial sources (see Table of Materials).
1. Generation of the hdIN precursors
NOTE: All the steps in this section are done in a cell culture hood. The hESCs were maintained as feeder-free cells on coated plates using a stem cell culture medium and passaged as colonies.
<ol>
	<li>Perform forward programming of the hESCs to hdIN precursors (Figure 1). 
	NOTE: The forward programming approach is based on the procedure described in Gonzalez-Ramos et al.8 and Yang et al.9.
	<ol>
		<li>Transduce the hESCs with lentiviral vectors containing the Tet-On system in a fresh stem cell culture medium containing 10 &#181;M of ROCK inhibitor (RI) (see Table of Materials). Keep at 37 &#176;C in the incubator.</li>
		<li>Change the medium to a fresh stem cell culture medium 16 h after transduction and maintain the transduced cells in the same way as the non-transduced hESCs.</li>
		<li>When confluent, detach the hESCs as single cells using a cell detachment solution (see Table of Materials) and plate them in coated 6-well plates at a density of 3 x 105 cells/well in stem cell culture medium containing 10 &#181;M of RI on day in vitro (DIV) &#8722;1.</li>
		<li>At 1 DIV, replace the culturing medium with N2 medium containing 2 g/L of doxycycline (DOX, see Table of Materials). 
		NOTE: DOX is used to induce transgene expression of Ascl1 and Dlx29, from 1 DIV to 7 DIV, and then continues in vivo by its addition to the drinking water25.</li>
		<li>At 2 DIV, an antibiotic resistance selection period starts that will last until 5 DIV. Add puromycin (puro) and hygromycin (hygro) to the fresh medium (see Table of Materials). Change the medium on 2 DIV, 4 DIV, and 5 DIV. 
		NOTE: Optimize the concentrations of puro and hygro before the differentiation protocol using transduced and non-transduced hESCs to ensure the survival of only the cells carrying the antibiotic resistance cassettes. In this protocol, 0.5 &#181;g/mL of puro and 750 &#181;g/mL of hygro were used.</li>
		<li>At 5 DIV, the antibiotic selection period ends. Change to N2 medium supplemented with 2 g/L of DOX and 4 &#181;M of cytosine &#946;-D-arabinofuranoside (Ara-C, see Table of Materials).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64272/64272fig01.jpg" /> 
Figure 1: Generation of hdIN precursors from hESCs by overexpressing Ascl1 and Dlx2. (A) Schematics of the differentiation protocol used for the generation of hdIN precursors. (B-E) Immunocytochemistry of hdIN precursors at 7 DIV for (B) the neuronal marker MAP2, (C) the proliferative marker Ki67, (D) general nuclear staining, and (E) a merge of the previous markers. Scale bar: 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64272/64272fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Preparation of the single-cell suspension for transplantation
NOTE: All the steps in this section are done in the cell culture hood. On 7 DIV, hdIN precursors are dissociated and used for transplantation.
<ol>
	<li>Perform detachment of the cells following the steps below.
	<ol>
		<li>Remove the medium and carefully rinse the cells with DPBS&#160;without calcium and magnesium.</li>
		<li>Add 400 &#181;L of the cell detachment solution (see Table of Materials) to each well of the 6-well plate. 
		NOTE: Ensure the coverage of the whole surface by the solution.</li>
		<li>Incubate for 2-3 min at 37 &#176;C in the incubator until the border of the cells starts to look shiny,&#160;indicating enzymatic degradation of cell surface proteins and detachment from the well surface. 
		NOTE: To increase cell survival, do not wait too long when all the cells are completely detached and floating around.</li>
		<li>Then, add 600 &#181;L of fresh N2 medium to each well of the 6-well plate to stop the enzyme, and mechanically with the pipette, help to detach the cells to obtain a single-cell suspension.</li>
		<li>Transfer the cell suspension to a plastic&#160;tube (15 mL) and centrifuge at 180 x g for 4 min at room temperature (RT).</li>
	</ol>
	</li>
	<li>Perform resuspension of the cells.
	<ol>
		<li>Discard the supernatant using a vacuum system (carefully, without disturbing the pellet) and resuspend the pellet in the transplantation medium containing 10 &#181;M of RI, 1 &#181;g/mL of DNase, and 2 &#181;g/&#181;L of DOX.</li>
		<li>Count the total cells in the suspension using a counting chamber and a manual cell counter and adjust the volume to a final concentration of 100,000 cells/&#181;L.</li>
		<li>Keep the cell suspension in a closed tube on ice until transplantation for a maximum of 4 h. 
		NOTE: The transplantation has to be performed on the same day as the preparation of the cell suspension (7 DIV).</li>
	</ol>
	</li>
</ol>
3. Intrahippocampal cell transplantation
NOTE: All the steps in this section are performed outside the cell culture hood in the animal facility. Early postnatal transplantation of cells into the brain was performed on P2, considering P0 the day of birth.
<ol>
	<li>Prepare for the transplantation experiment following the steps below.
	<ol>
		<li>Autoclave all the surgical material or, if not possible, sterilize with another approved method.</li>
		<li>Mount the injecting syringe in the holder without any glass capillary. Rinse the needle with water. 
		NOTE: The needle must be 33 G.</li>
		<li>Curve 90&#176; the tip of a 30 G insulin needle.</li>
		<li>Create a homemade Play-Doh-like stage for positioning the pup with the head flat (Figure 2A).</li>
		<li>Fetch the mice cage with the mother and pups before preparing anything for the surgery to allow them to acclimatize to the new environment.</li>
		<li>Prepare a tray with wet ice and an empty cage with some paper for the isoflurane.</li>
	</ol>
	</li>
	<li>Anesthetize the mouse pup.
	<ol>
		<li>Soak a piece of paper with isoflurane (see Table of Materials) and place it inside the empty cage. Carefully take one pup and place it into the cage with the paper soaked in isoflurane. 
		NOTE: Never leave less than three pups with the mum. Do not exceed 20-30 s of isoflurane anesthesia, or the pup may die.</li>
		<li>Verify the effect of the anesthesia by movement cessation.</li>
		<li>Immediately after anesthesia, place the pup onto a wet tissue on the surface of wet ice. Keep the animal on ice for 3 min until the upper limbs become whiteish (Figure 2B, blue arrow). This usually takes 2-3 min.</li>
		<li>Use this time for loading the syringe with the cell suspension. Resuspend the cells carefully with a pipette before.</li>
		<li>Place the pup on the stereotaxic frame. Use the ear bars in the opposite direction (Figure 2A, magenta arrows). 
		NOTE: The back side of the ear bars is less pointy, and as P2 pups do not have ears, use the flatter side to avoid hurting the animal.</li>
		<li>From the side, check if the head is straight. The head must be flat; use the Play-Doh-like stage to adjust for that. 
		NOTE: Pups at this age have not yet opened their eyes, so no additional step in this regard needs to be done. Pups do not have fur at this age, so no shaving of the area is required. No specific pain treatment is given as it is not an open incision to the skin, and no suturing is involved.</li>
		<li>Keep the pup covered on ice (on top of tissue paper) for the entire duration of the surgery. 
		NOTE: Do not place the ice in direct contact with the pup&#39;s skin.</li>
	</ol>
	</li>
	<li>Perform the injection.
	<ol>
		<li>Clean the surface of the skin using a soft tissue soaked in ethanol.</li>
		<li>Identify lambda and set the coordinates to zero on the digital display console of the stereotaxic instrument (Figure 2C, yellow dashed line). Sagittal and lambdoid sutures are easily visible by the eye, since they are vascularized, as red lines. 
		NOTE: If experiencing difficulties visualizing lambda, place a small piece of ice on the skin and wait a couple of minutes for it to cool down. Then, remove the piece of ice, and the subtle whitening of the skin will allow you to visualize.</li>
		<li>Relocate the injection needle to the desired coordinates. In the described protocol, the targeted region was the hippocampus, and the coordinates were as follows: anterior-posterior (AP) +0.85 and medio-lateral (ML) +1.35.</li>
		<li>Use a 90&#176; bent insulin needle to penetrate the skull and create a tiny hole.</li>
		<li>Bring down the injection needle until it has crossed the skull, and zero the dorso-ventral (DV) coordinates. Lower the needle until the desired DV coordinates. In the described protocol, the coordinates were DV &#8722;1.1.</li>
		<li>Inject according to the predetermined time intervals. 
		NOTE: In the described protocol, the exact timings were the following: (i) after going down to the DV coordinate, wait 3 min, (ii) inject 1 &#181;L of volume for 5 min, and (iii) after all the volume has been injected, wait 3 min.</li>
		<li>Retract the needle slowly.</li>
	</ol>
	</li>
	<li>End the procedure. 
	NOTE: The surgery cannot last more than 15 min to ensure the pup&#39;s survival and avoid any damage derived from the anesthesia by hypothermia.
	<ol>
		<li>Warm up the pup with the hands until it starts moving before giving it back to the mother.</li>
		<li>Give DOX at a concentration of 1 mg/mL in 0.5% sucrose solution as drinking water for at least 2 days before and 3 weeks post transplantation (PT) to continue the cell differentiation in vivo.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64272/64272fig02.jpg" /> 
Figure 2: Stereotaxic transplantation in newborn mice pups at P2. (A) A Play-Doh-like stage for holding the pup&#39;s body in position and inverted ear bars (magenta arrows). (B) White front paw (blue arrow) indicative of the reduced blood flow in that area so that the pup is experiencing anesthesia by hypothermia. (C) Overview of the setup with the pup already covered by ice over the soft tissue paper. (c#) Closed-zoom of the head of the pup, with the injection needle already inserted into the brain (yellow dashed line indicating lambda and lambdoid sutures). This figure is adapted from Gonzalez Ramos et al.27. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64272/64272fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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A., Lundberg, C., Bjorklund, A., Wictorin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+human+neural+progenitor+cells+into+the+neonatal+rat+brain:+Extensive+migration+and+differentiation+with+long-distance+axonal+projections.">Transplantation of human neural progenitor cells into the neonatal rat brain: Extensive migration and differentiation with long-distance axonal projections.</a> Experimental Neurology. 173 (1), 1-21 (2002).</li><li>Fainstein, N., Ben-Hur, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+region-dependent+rejection+of+neural+precursor+cell+transplants.">Brain region-dependent rejection of neural precursor cell transplants.</a> Frontiers in Molecular Neuroscience. 11, 136 (2018).</li><li>Denham, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurons+derived+from+human+embryonic+stem+cells+extend+long-distance+axonal+projections+through+growth+along+host+white+matter+tracts+after+intra-cerebral+transplantation.">Neurons derived from human embryonic stem cells extend long-distance axonal projections through growth along host white matter tracts after intra-cerebral transplantation.</a> Frontiers in Cellular Neuroscience. 6, 11 (2012).</li><li>Miguez, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+progressive+degeneration+of+Huntington's+disease+patient-derived+neurons+reveals+human-specific+pathological+phenotypes.">In vivo progressive degeneration of Huntington's disease patient-derived neurons reveals human-specific pathological phenotypes.</a> bioRxiv. , (2022).</li></ol>

The authors declare no competing interests.

The present protocol describes a robust, fast, simple, and widely accessible methodology to generate hdIN precursors in vitro and its use as early interventional cell therapy in preclinical models of neurodevelopmental disorders.
Even though some of the characteristic phenotypes of neurodevelopmental disorders arise during adolescence or adulthood, pathophysiological alterations are already present during early development. For this reason, early intervention would be highly warranted for achieving beneficial effects by acting in critical brain developmental periods before symptomatology or clinical manifestation. In the future, genetic screening and the development of biomarkers will afford prophylactic or pre-symptomatic treatment, representing a game changer for those patients. Therefore, hdIN precursors were transplanted early after birth in the Cntnap2 KO mouse model when epileptogenic changes in the neuronal network might be ongoing28 and at a timepoint at which cellular alterations have been described in this animal model29. It is important, however, to consider the potential pitfalls of age extrapolation and the timing of certain processes in the mouse versus the human brain.
Focusing on the procedure itself, the differentiation protocol presented here is based on the use of transcription factors, which allow for fast and highly efficient programming of stem cells compared with other protocols elsewhere based on small molecules10,30. A potential drawback of this approach could be the requirement for lentiviral vectors, which carries a risk of insertional mutagenesis. Two critical steps in the protocol are the addition of the antibiotics and the anti-mitotic agent to the medium to select for cells expressing the transcription factors and eliminate proliferative cells, avoiding the risk of teratoma formation, respectively. Although only hdIN precursors were tested in this study, the procedure is expected to be feasible with other cell sources and programming/differentiation protocols. Nevertheless, other neuronal subtypes and/or models should be validated.
The hdIN precursor&#39;s age for transplantation, 7 DIV, was decided based on (i) the absence of proliferative cells, assessed by immunoreactivity against Ki67, (ii) together with the previously reported observation of decreases in the expression of pluripotency genes such as POU5F1 and the appearance of the neuronal marker MAP2 and the interneuron marker GAD1 at that timepoint8. However, the original work describing this protocol performed transplantations at 14 DIV after DOX withdrawal9. This raises questions about whether DOX in the mother&#39;s drinking water can reach cells grafted into the brains of nursing pups via the milk, or if 7 DIV of DOX induction is enough to establish the GABAergic fate. Although Yang et al. identified 14 days of DOX as sufficient to generate stable neuronal cells in vitro9, Gonzalez-Ramos et al.8 detected GAD1 gene expression already at 7 DIV, indicating that the downstream activation of GAD67 by Ascl1 and Dlx2 has already occurred at this time point. Hence, patterning has begun at 7 DIV and might be less dependent on the DOX treatment. Moreover, evidence in rodents and humans indicates the presence of DOX in breast milk31,32, and the results presented here show that grafted hdINs were immunoreactive for Ascl1 at 2 weeks and 2 months PT and interneuron markers later on at 9 months PT. Within the grafted population, besides PV and SST positive neurons, other markers for subpopulations of interneurons were also found in lower amounts, such as calretinin (CR) and calbindin (CB).
A challenging aspect of this procedure is the coordination of the timings for both differentiation and the age of the pups. Usually, mouse gestation takes 21 days after setting up the mating cage, albeit this can vary sometimes. This scenario does not occur when performing cell transplantations in adult rodents when everything can be carefully planned and arranged. Nevertheless, this can be easily mitigated by setting up two to three mating cages with a 2 day interval or two to three differentiation batches with a 2 day time-lapse from each other.
Although the mice used in this study were neither immunodeficient nor immunosuppressed, the transplanted cells survived up to 9 months in vivo, and markers of immune reaction against xenogeneic cells or local inflammation were not observed at either P14 or 2 months PT. Immune rejection of grafted xenogeneic cells is triggered against MHC/peptides, and the key cellular mediators of graft rejection are T lymphocytes and microglial cells33,34. Therefore, immunoreactivity to markers of T cells, as well as reactive microglia, was explored. No signs of immune rejection of the grafted cells in the host tissue were detected either by levels of reactive microglia or by the presence of T lymphocytes in WT mice at P14 or 2 months. Moreover, no local inflammation was observed based on the assessed levels of astrogliosis and inflammatory cytokines. This outcome could partly be dependent on neonatal immune tolerance35,36,37, observed by other cell identities, locations, and animal models35,38. Englund et al. identified regional differences in the outcome of the grafted cells in terms of migration and maturation, including the observation of grafted cells in the adjacent white matter35.
Finally, a greater dispersion of the grafted cells within the hippocampus was observed compared to other studies transplanting into adult rodents, where hdINs remained as a grafted core25. This dispersion also differed from results observed previously by Yang et al.9, which could be explained in this case by the age of the cells at the time of transplantation.

The establishment, maturation, and refinement of neuronal networks occur during the perinatal and early postnatal period and represe a&#160;crucial time windows for brain development1. From an after-birth exuberance of connectivity, the brain evolves to a fine-tuning of connections that extend until adolescence2. Consequently, alterations in genes expressed during these periods, as well as external factors or insults, define an individual&#39;s predisposition to multiple neurodevelopmental disorders. Impairments in cognition and motor function unfold over time, and pharmacological treatments are limited, the majority targeting symptoms with the risk of severe side effects.
Dysfunction in gamma-aminobutyric acid (GABA)ergic inhibition has been shown to be a major contributor to the underlying cause of various neurodevelopmental disorders3, such as fragile X syndrome, Angelman syndrome, epilepsy, schizophrenia, and autism. GABA is the major inhibitory transmitter of the central nervous system and is instrumental for maintaining excitatory/inhibitory (E/I) balance, the synchronization of neuronal firing, and computation. GABAergic interneurons are a heterogeneous population of neurons, with increasing functional intricacy in more complex brain regions4 and with evolution5,6. Considering the limited endogenous regenerative capacity of the human brain and the implication of interneuron dysfunction in several neurological disorders, the transplantation of GABAergic interneurons may be a promising therapeutic avenue to explore. Along this line, human stem cell-derived GABAergic interneuron-like cells (hdINs) seem to be the most translational and viable source for this purpose compared to rodent allogenic neuronal precursors or other sources used elsewhere7. Protocols for generating GABAergic neurons from diverse cell sources are available8,9,10,11,12,13, but more knowledge is required on how hdINs mature, integrate, and function over time in a developing pathologic brain. Several studies have identified alterations in genes active during cortical patterning, establishing neuronal connectivity14, and tuning physiological E/I balance15. Neonatal transplantation of hdINs into mouse models with corresponding genetic perturbations allows us to follow the interplay between host and graft, which is knowledge necessary to determine potential therapeutic strategies.
Immunomodulation is commonly and successfully used in xenograft transplantations to avoid triggering a host immune response and rejection16. However, the administration of immunosuppressive drugs, such as cyclosporine A, causes renal toxicity after chronic administration, is labor-intensive because of the need for daily intraperitoneal injections to achieve stable systemic concentrations, causing animal stress17, and has off-target effects that can interact with pathology18. In addition, compromising the immune system has been shown to change behavioral phenotypes19, with alterations in the corresponding neuroanatomical regions20. Transplantation in the first week of life has been shown to allow for adaptation to transplanted cells21,22, while others have reported initial survival followed by rejection of the grafts within the first postnatal month23,24.
This protocol describes procedures from hdIN generation to cell transplantation in neonatal mice resulting in long-term graft survival and allowing for the investigation of the neuronal specificity, synaptic integration, function, and therapeutic potential of transplanted human interneurons during physiological and pathological development.

<table><tbody><tr><td>30 G needle</td><td>B Braun</td><td>4656300</td><td></td></tr><tr><td>33 G needle for Hamilton syringe</td><td>Hamilton</td><td>7762-06</td><td></td></tr><tr><td>4-well plates</td><td>Thermo Scientific</td><td>176740</td><td></td></tr><tr><td>Accutase</td><td>STEMCELL Technologies</td><td>7920</td><td>Cell detachment solution use for splitting cells (hESC and hdIN precursors)</td></tr><tr><td>Adjustable volume pipettes 10, 20, 200, 1000 &amp;micro;L</td><td></td><td></td><td></td></tr><tr><td>Alexa Fluor Plus 488/555/647</td><td>Thermo Fisher</td><td></td><td>1:1000</td></tr><tr><td>Anti-CD68 (Rat)</td><td>Bio-Rad</td><td>MCA1957</td><td>1:200</td></tr><tr><td>Anti-CD8 (Rabbit)</td><td>Abcam</td><td>203035</td><td>1:200</td></tr><tr><td>Anti-Galectin 3 (Goat)</td><td>R&amp;amp;D systems</td><td>AF1197</td><td>1:500</td></tr><tr><td>Anti-GFAP (Guiena Pig)</td><td>Synaptic systems</td><td>173004</td><td>1:500</td></tr><tr><td>Anti-Iba1 (Rabbit)</td><td>WAKO</td><td>19119741</td><td>1:500</td></tr><tr><td>Anti-IL1 (Goat)</td><td>Santa Cruz Biotech</td><td>SC-106</td><td>1:400</td></tr><tr><td>Anti-Ki67</td><td>Abcam</td><td>ab16667</td><td>1:250</td></tr><tr><td>Anti-Ki67 (Rabbit)</td><td>Novocastra</td><td>NCL-Ki67p</td><td>1:250</td></tr><tr><td>Anti-MAP2 (Chicken)</td><td>Abcam</td><td>ab5392</td><td>1:2000</td></tr><tr><td>Anti-Mash1 (Ascl1)</td><td>Abcam</td><td>ab74065</td><td>1:1000</td></tr><tr><td>Anti-Parvalbumin (Rabbit)</td><td>Swant</td><td>PV 27</td><td>1:5000</td></tr><tr><td>Anti-Somatostatin (Rat)</td><td>Millipore</td><td>MAB354</td><td>1:150</td></tr><tr><td>Anti-STEM121 (Mouse)</td><td>Takara Bio</td><td>Y40410</td><td>1:400</td></tr><tr><td>Avidin/Biotin Blocking Kit</td><td>VECTOR Laboratories</td><td>SP-2001</td><td></td></tr><tr><td>B6.129(Cg)-Cntnap2tm1Pele/J</td><td>Jackson Laboratory</td><td>17482</td><td>Animal model</td></tr><tr><td>Biotinylated Horse anti-Mouse</td><td>VECTOR Laboratories</td><td>BA-2001</td><td>1:200</td></tr><tr><td>Burker Chamber</td><td>Thermo Fisher Scientific</td><td>10628431</td><td></td></tr><tr><td>C57BL/6J</td><td>Janvier Labs</td><td></td><td>Animal model</td></tr><tr><td>Centrifuge</td><td></td><td></td><td>For 15 mL tubes</td></tr><tr><td>Confocal microscope</td><td>Nikon&amp;nbsp;</td><td>Confocal A1RHD microscope</td><td></td></tr><tr><td>Costar 6-well Clear TC-treated&amp;nbsp;</td><td>Corning</td><td>3516</td><td></td></tr><tr><td>Cy3 Streptavidin</td><td>Jackson ImmunoResearch</td><td>016-160-084</td><td>1:200</td></tr><tr><td>Cytosine &amp;beta;-D-arabinofuranoside (AraC)</td><td>Sigma</td><td>C1768</td><td>4 &amp;micro;M</td></tr><tr><td>DAB Substrate Kit, Peroxidase (With Nickel)</td><td>VECTOR Laboratories</td><td>SK-4100</td><td></td></tr><tr><td>Digital Stereotax</td><td>KOPF</td><td>Model 940</td><td></td></tr><tr><td>DMEM/F12</td><td>Thermo Fisher Scientific</td><td>11320082</td><td>Use for the N2 medium</td></tr><tr><td>DNase I Solution</td><td>STEMCELL Technologies</td><td>7900</td><td>1 &amp;micro;g/mL</td></tr><tr><td>Doxycyclin</td><td>Sigma-Aldrich</td><td>D9891</td><td>2 &amp;micro;g/mL</td></tr><tr><td>DPBS -/-</td><td>Gibco</td><td>14190144</td><td></td></tr><tr><td>Epifluorescence microscope</td><td>Olympus</td><td>BX51 Microscope</td><td></td></tr><tr><td>Ethanol</td><td>Solveco</td><td></td><td>70%, 95%, 99.8%</td></tr><tr><td>FUW-rTA</td><td>Addgene</td><td>20342</td><td>Lentiviral vector</td></tr><tr><td>FUW-TetO-Ascl1-T2A-puromycin</td><td>Addgene</td><td>97329</td><td>Lentiviral vector</td></tr><tr><td>FUW-TetO-Dlx2-IRES-hygromycin</td><td>Addgene</td><td>97330</td><td>Lentiviral vector</td></tr><tr><td>H1 (WA01) ESC</td><td>WiCell</td><td>WA01</td><td>Human embryonic stem cell line under a MTA agreement</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>18304</td><td></td></tr><tr><td>Hamilton Syringe</td><td>Hamilton</td><td>7634-01</td><td>5 &amp;micro;L</td></tr><tr><td>HBSS</td><td>Gibco</td><td>14175095</td><td>No calcium, No magnesium - Transplantation medium</td></tr><tr><td>Hoechst 33342</td><td>Invitrogen</td><td>H3570</td><td>1:1000</td></tr><tr><td>Hygromycin B</td><td>Gibco (Invitrogen)</td><td>10687010</td><td></td></tr><tr><td>Incubator</td><td></td><td></td><td>5% CO&lt;sub&gt;2&lt;/sub&gt;, 37 &amp;deg;C</td></tr><tr><td>Isoflurane Baxter</td><td>Apoteket AB</td><td></td><td></td></tr><tr><td>Manual cell counter</td><td>VWR</td><td>720-1984</td><td></td></tr><tr><td>Matrigel hESC-Qualified Matrix, LDEV-free</td><td>Corning</td><td>354277</td><td>For the coating</td></tr><tr><td>Methanol</td><td>Merck Millipore</td><td>1060091000</td><td></td></tr><tr><td>Microscope Coverslips 24 x 60 mm</td><td>Thermo Scientific</td><td>BBAD02400500#A113MNZ#0##</td><td></td></tr><tr><td>Microscope Slides</td><td>VWR</td><td>631-1551</td><td></td></tr><tr><td>Microscope Software</td><td>Olympus</td><td>CellSens</td><td></td></tr><tr><td>Mounting media</td><td>Merck</td><td>10981</td><td>PVA-Dabco&amp;nbsp;</td></tr><tr><td>Mouse adaptor to stereotax</td><td>RWD</td><td>68030</td><td></td></tr><tr><td>mTeSR1</td><td>STEMCELL Technologies</td><td>85850</td><td>Kit Basal Medium and 5X Supplement - Stem cell culture medium</td></tr><tr><td>N2 supplement</td><td>Gibco</td><td>17502048</td><td></td></tr><tr><td>NaOH</td><td>Sigma-Aldrich</td><td>S8045</td><td>1M</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P0781</td><td></td></tr><tr><td>Pertex</td><td>HistoLab</td><td>811</td><td></td></tr><tr><td>Pipet Filler</td><td></td><td></td><td></td></tr><tr><td>Play-Doh</td><td></td><td></td><td></td></tr><tr><td>Puromycin (Dihydrochloride)</td><td>Gibco</td><td>A1113803</td><td></td></tr><tr><td>Round cover glasses thickness No. 1.5H (tol. &amp;plusmn; 5 &amp;mu;m) 13 mm &amp;Oslash;</td><td>Marienfeld</td><td>MARI0117530</td><td>For immunocytochemistry</td></tr><tr><td>Serum</td><td>Thermo Fisher</td><td></td><td>Goat, Donkey, Horse</td></tr><tr><td>Sterile pipette tips</td><td></td><td></td><td>For volumes 0.1-1000 &amp;micro;L</td></tr><tr><td>Sterile serological pipettes</td><td></td><td></td><td>5, 10, 25 mL</td></tr><tr><td>Sterile water Braun</td><td>B Braun</td><td>3626873</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S8501</td><td>For 0.5% Sucrose solution</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Trypan Blue Solution</td><td>Gibco</td><td>15250061</td><td></td></tr><tr><td>Tubes</td><td>Sarstedt</td><td></td><td>15 ml, Eppendorf 1.5 mL</td></tr><tr><td>Tweezer</td><td>VWR</td><td></td><td></td></tr><tr><td>Ultra pure water</td><td></td><td></td><td>MilliQ Water System</td></tr><tr><td>Xylene</td><td>VWR</td><td>28973.363</td><td></td></tr><tr><td>Y-27632 (ROCK inhibitor)</td><td>STEMCELL Technologies</td><td>72304</td><td>10 &amp;micro;M</td></tr></tbody></table>

transplantation of human stem cell-derived gabaergic neurons into the early postnatal mouse hippocampus to mitigate neurodevelopmental disorders

A reduced number or dysfunction of inhibitory interneurons is a common contributor to neurodevelopmental disorders. Therefore, cell therapy using interneurons to replace or mitigate the effects of altered neuronal circuits is an attractive therapeutic avenue. To this end, more knowledge is needed about how human stem cell-derived GABAergic interneuron-like cells (hdINs) mature, integrate, and function over time in the host circuitry. Of particular importance in neurodevelopmental disorders is a better understanding of whether these processes in transplanted cells are affected by an evolving and maturing host brain. The present protocol describes a fast and highly efficient generation of hdINs from human embryonic stem cells based on the transgenic expression of the transcription factors Ascl1 and Dlx2. These neuronal precursors are transplanted unilaterally, after 7 days in vitro, to the hippocampus of neonatal 2-day-old mice. The transplanted neurons disperse in the ipsi- and contralateral hippocampus of a mouse model of cortical dysplasia-focal epilepsy syndrome and survive for up to 9 months after transplantation. This approach allows for investigating the cellular identity, integration, functionality, and therapeutic potential of transplanted interneurons over an extended time in developing healthy and diseased brains.

Following the protocol presented here and illustrated in Figure 1A, hdIN precursors were not proliferative yet at 7 DIV as defined by (i) negative immunoreactivity for the cell cycle marker Ki67 and (ii) expressing neuronal markers such as microtubule-associated protein 2 (MAP2) (Figure 1B-E). This characterization was performed on leftover cells replated for 24 h after having undergone all the procedure steps. In addition, the gene expression analysis published previously indicated that a rapid transition from the pluripotent state to a neuronal phenotype occurs around 4 DIV and 7 DIV8. Overall, these results confirmed the presence of postmitotic cells and the absence of risk for teratoma formation.
Next, the hdIN precursors&#39; survival after early postnatal transplantation into the hippocampus of wild-type (WT) mice was tested by immunohistochemistry against the human cytoplasmatic marker STEM121. The hdIN precursors were transplanted into the right dorsal hippocampus of na&#239;ve&#160;immunocompetent mice at P2, which were then sacrificed at P14 and 2 months PT. Grafted cells were found across the whole dorsal hippocampus, as well as dispersed through the corpus callosum and the contralateral hippocampus, at both time points. Moreover, at both time points, grafted hdINs expressed Ascl1, one of the induction transcription factors (Supplementary Figure 1), and were not proliferative, as indicated by the absence of Ki67 expression (Supplementary Figure 2).
Importantly, no immune reaction or local inflammation against the transplanted cells was found either at P14 or 2 months PT, as assessed by the absence of reactive microglia identified using Iba1, CD68, and galectin-3 (Gal3) (Figure 3), the extent of astrogliosis determined by the glial fibrillary acidic protein (GFAP) and inflammatory cytokines such as interleukine-1 (IL-1), and the absence of cytotoxic T lymphocytes (CD8) (Figure 4).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64272/64272fig03.jpg" /> 
Figure 3: hdINs at P14 and 2 months PT into the hippocampus of newborn WT mice without triggering immune rejection from the host tissue. Immunofluorescence for Iba1, CD68, and Gal3 markers in brain tissue from (A-D) the proximity of an ischemic core area in an electrocoagulation stroke mouse model (positive control, Ctrl +), (E-H) negative control animals (Ctrl-) at 2 months and (M-P) P14, and (I-L) animals that have undergone cell transplantation at 2 months and (Q-T) P14. The white arrows indicate some examples of blood vessels visible at all channels due to autofluorescence. Abbreviations: Ctx = cortex; Hip = hippocampus; DG = dentate gyrus; CA1 = cornu ammonis 1. Scale bar: 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64272/64272fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64272/64272fig04.jpg" /> 
Figure 4: hdINs at 2 months PT into the hippocampus of newborn WT mice without triggering immune rejection from the host tissue. Immunofluorescence for IL1, GFAP, and CD8 markers in brain tissue from (A-D) the proximity of an ischemic core area in an electrocoagulation stroke mouse model (positive control, Ctrl +), (E-H)&#160;negative control animals (Ctrl-) at 2 months, and (I-L)animals that have undergone cell transplantation at 2 months. Abbreviations: Ctx = cortex; Hip = hippocampus; DG = dentate gyrus. Scale bar: 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64272/64272fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Similarly, hdIN precursors were also transplanted into the hippocampus of Cntnap2 KO mice, a model for autism spectrum disorder and cortical dysplasia-focal epilepsy syndrome. In the Cntnap2 KO mice, indeed, the hdINs survived up to 9 months PT and were localized at the injection site, although they were also dispersed across the ipsilateral and even the contralateral hippocampus as observed in the WT mice (Figure 5). Moreover, most of the grafted hdINs were immunoreactive for interneuron markers, as expected from previous results in vitro8,26 and in adult rodents in vivo25.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64272/64272fig05.jpg" /> 
Figure 5: Grafted hdINs in the hippocampus of Cntnap2 KO mice at 9 months PT. (A) Immunochemistry against the cytoplasmatic human marker STEM121 in cell-transplanted (left) and sham (right) mice. (a1 and a2) Magnified images of STEM121 positive cells. (B) Immunofluorescence for STEM121 (magenta) and the interneuron markers parvalbumin (PV) and somatostatin (SST). White arrows indicate double positive cells for STEM121 and the respective interneuron marker. (C) Orthogonal view of a grafted hdIN immunoreactive for STEM121 and PV. Scale bar: 200 &#181;m (A and B), 100 &#181;m (a1 and a2), 20 &#181;m (small square magnification in a1 and a2, and C). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64272/64272fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Grafted hdINs at 2 months PT into the hippocampus of newborn WT mice expressing Ascl1. Immunofluorescence against Ascl1 and the cytoplasmatic human marker STEM121 at the (A) CA3 and (B) DG in cell-transplanted WT mice. (a) Magnified image of a STEM121 positive cell. The white arrows indicate double-positive cells for STEM121 and Ascl1. Scale bar: 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64272/Supplementary Figure 1_vertical_.eps" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Post-mitotic grafted hdINs at 2 months PT into the hippocampus of newborn WT mice. Immunofluorescence against the proliferative marker Ki67 and the cytoplasmatic human marker STEM121 in (A) na&#239;ve and (B) cell-transplanted WT mice. (b) Magnified image of a STEM121 positive cell. The yellow arrow indicates a cell positive for Ki67 and negative for STEM121. The white arrow indicates cells positive for STEM121 and negative for Ki67. The white asterisk points out a lateral ventricle. Scale bar: 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64272/Supplementary Figure 2_vertical_.eps" target="_blank">Please click here to download this File.</a>

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Transplantation of Human Stem Cell-Derived GABAergic Neurons into the Early Postnatal Mouse Hippocampus to Mitigate Neurodevelopmental Disorders

Transplantation of human pluripotent stem cell-derived GABAergic neurons generated by neuronal programming could be a potential treatment approach for neurodevelopmental disorders. This protocol describes the generation and transplantation of human stem cell-derived GABAergic neuronal precursors into the brains of neonatal mice, allowing long-term investigation of grafted neurons and evaluation of their therapeutic potential.

A reduced number or dysfunction of inhibitory interneurons is a common contributor to neurodevelopmental disorders. Therefore, cell therapy using ...

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2.3K Views.  Lund University Hospital. Deze aanpak maakt het mogelijk om cellulaire identiteit, integratie en functionaliteit van getransplanteerde interneuronen gedurende langere tijd te onderzoeken bij het ontwikkelen van gezonde en zieke hersenen. Het protocol beschrijft een snelle en zeer efficiënte generatie van menselijke stamcel-afgeleide GABAerge interneuronachtige voorlopers die overleven en rijpen in de hippocampus van naïeve en transgene muizen. Deze aanpak helpt ons het therapeutisch potentieel te evalueren van het g...

Video: Transplantation of Human Stem Cell-Derived GABAergic Neurons into the Early Postnatal Mouse Hippocampus to Mitigate Neurodevelopmental Disorders

Stem Cell Transplantation Strategies for the Restoration of Cognitive Dysfunction Caused by Cranial Radiotherapy

Radiotherapy often provides the only clinical recourse for those afflicted with primary or metastatic brain tumors. While beneficial, cranial irradiation can induce a progressive and debilitating decline in cognition that may, in part, be caused by the depletion of neural stem cells. Given the increased survival of patients diagnosed with brain cancer, quality of life in terms of cognitive health has become an increasing concern, especially in the absence of any satisfactory long-term treatments.
To address this serious health concern we have used stem cell replacement as a strategy to combat radiation-induced cognitive decline. Our model utilizes athymic nude rats subjected to cranial irradiation. The ionizing radiation is delivered as either whole brain or as a highly focused beam to the hippocampus via linear accelerator (LINAC) based stereotaxic radiosurgery. Two days following irradiation, human neural stem cells (hNSCs) were stereotaxically transplanted into the hippocampus. Rats were then assessed for changes in cognition, grafted cell survival and for the expression of differentiation-specific markers 1 and 4-months after irradiation. Our cognitive testing paradigms have demonstrated that animals engrafted with hNSCs exhibit significant improvements in cognitive function. Unbiased stereology reveals significant survival (10-40%) of the engrafted cells at 1 and 4-months after transplantation, dependent on the amount and type of cells grafted. Engrafted cells migrate extensively, differentiate along glial and neuronal lineages, and express a range of immature and mature phenotypic markers.
Our data demonstrate direct cognitive benefits derived from engrafted human stem cells, suggesting that this procedure may one day afford a promising strategy for the long-term functional restoration of cognition in individuals subjected to cranial radiotherapy. To promote the dissemination of the critical procedures necessary to replicate and extend our studies, we have provided written and visual documentation of several key steps in our experimental plan, with an emphasis on stereotaxic radiosurgey and transplantation.

Brain tumor patients routinely undergo cranial radiotherapy, and while beneficial, this treatment often results in debilitating cognitive dysfunction. This serious unresolved problem has at present, no clinical recourse, and has driven our efforts to devise stem cell based therapies for the recovery of radiation-induced cognitive decrements.

Radiotherapy often provides the only clinical recourse for those afflicted with primary or metastatic brain tumors. While beneficial, cranial irradiation ...

Medicine

Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the demyelinating disease Multiple Sclerosis (MS). We have shown that transplantation of NSCs into the spinal cords of sick mice results in a significant improvement in both remyelination and in clinical outcome. Cell replacement therapies for the treatment of chronic neurologic diseases are now a reality and in vivo models are vital in understanding the interactions between the engrafted cells and host tissue microenvironment. This presentation provides an adapted method for transplanting cells into the spinal cord of JHMV-infected mice. In brief, we provide a procedure for i) preparation of NSCs prior to transplant, ii) pre-operative care of mice, iii) exposure of the spinal cord via laminectomy, iv) stereotactic injection of NSCs, and iv) post-operative care.

The transplantation of mouse neural stem cells (NSCs) into the spinal cords of mice with established demyelination is detailed. The preparation of NSCs, the laminectomy of thoracic vertebra 9 (T9), and transplantation of NSCs is outlined along with the pre- and post-operative care of the mice.

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

Developmental Biology

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the roles of intrinsic vs. extrinsic mechanisms that regulate this differentiation process is a conundrum for all developmental neurobiologists. This issue is magnified for GABAergic interneurons, an incredibly heterogeneous cell population that is born from transient embryonic structures and undergo a protracted migratory phase to disperse throughout the telencephalon. To explore how different brain environments affect interneuron fate and maturation, we developed a protocol for harvesting fluorescently labeled immature interneuron precursors from specific brain regions in newborn mice (P0-P2). At this age, interneuron migration is nearly complete and these cells are residing in their final resting environments with relatively little synaptic integration. Following collection of single cell solutions via flow cytometry, these interneuron precursors are transplanted into P0-P2 wildtype postnatal pups. By performing both homotopic (e.g., cortex-to-cortex) or heterotopic (e.g., cortex-to-hippocampus) transplantations, one can assess how challenging immature interneurons in new brain environments affects their fate, maturation, and circuit integration. Brains can be harvested in adult mice and assayed with a wide variety of posthoc analysis on grafted cells, including immunohistochemical, electrophysiological and transcriptional profiling. This general approach provides investigators with a strategy to assay how distinct brain environments can influence numerous aspects of neuron development and identify if specific neuronal characteristics are primarily driven by hardwired genetic programs or environmental cues.

Challenging young neurons in new brain regions can reveal important insights into how the environment sculpts neuronal fate and maturation. This protocol describes a procedure to harvest interneuron precursors from specific brain regions and transplant them either homotopically or heterotopically into the brain of postnatal pups.

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the ...

Electrophoretic Delivery of &#x3B3;-aminobutyric Acid (GABA) into Epileptic Focus Prevents Seizures in Mice

Epilepsy is a group of neurological disorders which affects millions of people worldwide. Although treatment with medication is helpful in 70% of the cases, serious side effects affect the quality of life of patients. Moreover, a high percentage of epileptic patients are drug resistant; in their case, neurosurgery or neurostimulation are necessary. Therefore, the major goal of epilepsy research is to discover new therapies which are either capable of curing epilepsy without side effects or preventing recurrent seizures in drug-resistant patients. Neuroengineering provides new approaches by using novel strategies and technologies to find better solutions to cure epileptic patients at risk.
As a demonstration of a novel experimental protocol in an acute mouse model of epilepsy, a direct in situ electrophoretic drug delivery system is used. Namely, a neural probe incorporating a microfluidic ion pump (&#181;FIP) for on-demand drug delivery and simultaneous recording of local neural activity is implanted and demonstrated to be capable of controlling 4-aminopyridine-induced (4AP-induced) seizure-like event (SLE) activity. The &#947;-aminobutyric acid (GABA) concentration is kept in the physiological range by the precise control of GABA delivery to reach an antiepileptic effect in the seizure focus but not to cause overinhibition-induced rebound bursts. The method allows both the detection of pathological activity and intervention to stop seizures by delivering inhibitory neurotransmitters directly to the epileptic focus with precise spatiotemporal control.
As a result of the developments to the experimental method, SLEs can be induced in a highly localized manner that allows seizure control by the precisely tuned GABA delivery at the seizure onset.

Electrophoretic Delivery of &#947;-aminobutyric Acid GABA into Epileptic Focus Prevents Seizures in Mice

The challenge of epilepsy research is to develop novel treatments for patients where classical therapy is inadequate. Using a new protocol&#8212;with the help of an implantable drug delivery system&#8212;we are able to control seizures in anesthetized mice by the electrophoretic delivery of GABA into the epileptic focus.

Epilepsy is a group of neurological disorders which affects millions of people worldwide. Although treatment with medication is helpful in 70% of the ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain functions. Most of our current understanding of microglia is based on experiments performed in the mouse. Human microglia differ from mouse microglia, and thus response and characteristics of mouse microglia may not always represent that of human microglia. Further, due to ethical and technical difficulties, research on human microglia is restricted to in vitro culture system, which does not capitulate in vivo characteristics of microglia. To overcome these issues, a simplified method to non-invasively transplant induced pluripotent stem cell-derived human microglia (iPSMG) into the immunocompetent mice brain via a transnasal route in combination with pharmacological depletion of endogenous microglia using a colony-stimulating factor 1 receptor (CSF1R) antagonist is developed. This protocol provides a way to non-invasively transplant cells into the mouse brain and may therefore be valuable for evaluating the in vivo role of human microglia in physiological and pathological brain functions.

The protocol presented here allows the transplantation of induced pluripotent stem cell-derived human microglia (iPSMG) into the brain via a transnasal route in immunocompetent mice. The method for the preparation and transnasal transplantation of cells and the administration of cytokine mixture for the maintenance of iPSMG is shown.

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain ...

Transplantation of Human Stem Cell-Derived GABAergic Neurons into the Early Postnatal Mouse Hippocampus to Mitigate Neurodevelopmental Disorders

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