Grant sponsors: This study was supported by JSPS KAKENHI 17K14961 (PB), 20K15899 (PB), JP18K06481 (YS), JP20KK0366 (YS), 20H05902 (SK), 20H05060 (SK), 19H04746 (SK), 21H04786 (SK), 21K19309 (SK), AMED-CREST (SK), CREST (SK), the Mitsubishi Science Foundation (SK), the Takeda Science Foundation (SK), and a Frontier Brain Science Grant from the University of Yamanashi (SK).

All animals used in this study were obtained, housed, cared for, and used in accordance with the &#34;Guiding Principles in the Care and Use of Animals in the Field of Physiologic Sciences&#34; published by the Physiologic Society of Japan20, and with the previous approval of the Animal Care Committee of the University of Yamanashi (Yamanashi, Japan).
1. Preparation of cell medium, transplantation medium, and anesthesia mixture
<ol>
	<li>Prepare the cell medium by adding 10% fetal bovine serum and 0.1% penicillin/streptomycin in DMEM.</li>
	<li>Prepare the transplantation medium by adding hCSF1 (250 ng/mL) and hTGF-&#946;1 (100 ng/mL) to the cell medium.</li>
	<li>Prepare the anesthesia mixture for intraperitoneal injection by mixing 0.45 mL of medetomidine hydrochloride, 1.2 mL of midazolam, and 1.5 mL of butorphanol tartrate in 11.8 mL of normal saline.</li>
</ol>
2. Preparation of iPSMG
NOTE: Frozen iPSMG (Table of Materials) were kept at -80 &#176;C until use.
<ol>
	<li>Thaw the frozen cells quickly in a 37 &#176;C water bath. Swirl the samples until all visible ice has melted.</li>
	<li>Add the thawed iPSMG to the culture media warmed to 37 &#176;C. Add 1 mL of thawed cells containing medium (1 &#215; 106 cells) to 10 mL of the culture media.</li>
	<li>Centrifuge the cells at 300 x g for 5 min to obtain a cell pellet.</li>
	<li>After centrifugation, remove all supernatant without disturbing the cell pellet. Complete removal of supernatant is desirable to reduce dilution of the cytokine concentration in the transplantation medium.</li>
	<li>Add the transplantation medium to obtain a cell concentration of 1 x 105 cells/&#181;L.</li>
	<li>Place the iPSMG on ice and immediately proceed to transplantation. 
	&#8203;NOTE: Preparation of iPSMG for transplantation is performed on a clean bench to avoid contamination.</li>
</ol>
3. Preparation of mouse for transnasal transplantation (Tsn)
<ol>
	<li>Feed the wild-type male mice (C57BL/6J, 8 weeks old) with PLX5622 containing diet for 7 days.</li>
	<li>At the end of the 7th day, cease the PLX diet and feed the mice with a normal diet till the end of study. 
	NOTE: PLX5622 containing diet is prepared by adding 1.2 g of PLX5622 in 1 kg of AIN-93G (Table of Materials).</li>
</ol>
4. Transnasal transplantation of cells
<ol>
	<li>24 h after the cessation of PLX feeding, weigh the mice and anesthetize them using an intraperitoneal injection of the anesthesia mixture (0.2 mL/20 g).</li>
	<li>After the mice are completely anesthetized as assessed by unresponsiveness to pedal withdrawal reflex (firm toe pinch), administer 2.5 &#181;L of hyaluronidase in PBS (100 U/mL) at 1 h before Tsn of iPSMG to each nostril twice using a 10 &#181;L pipette tip to increase the permeability of the nasal mucosa.</li>
	<li>After the application of hyaluronidase, place the mice in the supine position.</li>
	<li>Repeat step 4.2 10 min before the transnasal transplantation of iPSMG.</li>
	<li>Apply 2.5 &#181;L of cell suspension into one nostril of the mouse using a 10 &#181;L pipette tip.</li>
	<li>Place the mouse in the supine position for 5 min before administration of cell suspension to the other nostril.</li>
	<li>Repeat steps 4.5 and 4.6 four times, applying a total volume of 20 &#181;L per animal.</li>
	<li>Place the mouse in the supine position on a 37 &#176;C heat pad until recovery from anesthesia.</li>
	<li>At 48 h after the cessation of PLX feeding, repeat steps 4.1-4.7 on the same mice once again. 
	&#8203;NOTE: It should be noted that iPSMG are placed on ice during the transplantation.</li>
</ol>
5. Application of cytokines
<ol>
	<li>Anesthetize the mice by an intraperitoneal injection of anesthesia mixture (0.2 mL/20 g).</li>
	<li>Apply 2.5 &#181;L of the transplantation medium into one nostril of the mouse using a 10 &#181;L pipette tip.</li>
	<li>Place the mouse in the supine position for 5 min before administering the transplantation medium to the other nostril.</li>
	<li>Repeat steps 5.2 and 5.3 four times, applying a total volume of 20 &#181;L per animal. 
	NOTE: It should be noted that transnasal administration of transplantation medium (human cytokines) is required every 12 h for the viability of transplanted iPSMG till the end of the study.</li>
</ol>

<ol>
	<li>Hanisch, U. K., Kettenmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia:+active+sensor+and+versatile+effector+cells+in+the+normal+and+pathologic+brain.">Microglia: active sensor and versatile effector cells in the normal and pathologic brain.</a> Nature Neuroscience. 10 (11), 1387-1394 (2007).</li><li>Nimmerjahn, A., Kirchhoff, F., Helmchen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resting+microglial+cells+are+highly+dynamic+surveillants+of+brain+parenchyma+in+vivo.">Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo.</a> Science. 308 (5726), 1314-1318 (2005).</li><li>Paolicelli, R. C., 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=Synaptic+pruning+by+microglia+is+necessary+for+normal+brain+development.">Synaptic pruning by microglia is necessary for normal brain development.</a> Science. 333 (6048), 1456-1458 (2011).</li><li>Smith, A. M., Dragunow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+side+of+microglia.">The human side of microglia.</a> Trends in Neurosciences. 37 (3), 125-135 (2014).</li><li>Galatro, T. F., 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=Transcriptomic+analysis+of+purified+human+cortical+microglia+reveals+age-associated+changes.">Transcriptomic analysis of purified human cortical microglia reveals age-associated changes.</a> Nature Neuroscience. 20 (8), 1162-1171 (2017).</li><li>Gosselin, D., 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=An+environment-dependent+transcriptional+network+specifies+human+microglia+identity.">An environment-dependent transcriptional network specifies human microglia identity.</a> Science. 356 (6344), (2017).</li><li>Abud, E. 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=iPSC-derived+human+microglia-like+cells+to+study+neurological+diseases.">iPSC-derived human microglia-like cells to study neurological diseases.</a> Neuron. 94 (2), 278-293 (2017).</li><li>Brownjohn, P. W., 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=Functional+studies+of+missense+TREM2+mutations+in+human+stem+cell-derived+microglia.">Functional studies of missense TREM2 mutations in human stem cell-derived microglia.</a> Stem Cell Reports. 10 (4), 1294-1307 (2018).</li><li>Douvaras, P., 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=Directed+differentiation+of+human+pluripotent+stem+cells+to+microglia.">Directed differentiation of human pluripotent stem cells to microglia.</a> Stem Cell Reports. 8 (6), 1516-1524 (2017).</li><li>Haenseler, W., 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=A+highly+efficient+human+pluripotent+stem+cell+microglia+model+displays+a+neuronal-co-culture-specific+expression+profile+and+inflammatory+response.">A highly efficient human pluripotent stem cell microglia model displays a neuronal-co-culture-specific expression profile and inflammatory response.</a> Stem Cell Reports. 8 (6), 1727-1742 (2017).</li><li>Hasselmann, J., 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=Development+of+a+chimeric+model+to+study+and+manipulate+human+microglia+in+vivo.">Development of a chimeric model to study and manipulate human microglia in vivo.</a> Neuron. 103 (6), 1016-1033 (2019).</li><li>Mancuso, R., 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=Stem-cell-derived+human+microglia+transplanted+in+mouse+brain+to+study+human+disease.">Stem-cell-derived human microglia transplanted in mouse brain to study human disease.</a> Nature Neuroscience. 22 (12), 2111-2116 (2019).</li><li>Muffat, J., 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=Efficient+derivation+of+microglia-like+cells+from+human+pluripotent+stem+cells.">Efficient derivation of microglia-like cells from human pluripotent stem cells.</a> Nature Medicine. 22 (11), 1358-1367 (2016).</li><li>Pandya, H., 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=Differentiation+of+human+and+murine+induced+pluripotent+stem+cells+to+microglia-like+cells.">Differentiation of human and murine induced pluripotent stem cells to microglia-like cells.</a> Nature Neuroscience. 20 (5), 753-759 (2017).</li><li>Schetters, S. T. T., Gomez-Nicola, D., Garcia-Vallejo, J. J., Van Kooyk, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroinflammation:+Microglia+and+T+cells+get+ready+to+tango.">Neuroinflammation: Microglia and T cells get ready to tango.</a> Frontiers in Immunology. 8, 1905 (2017).</li><li>Sulzer, D., 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=T+cells+from+patients+with+Parkinson's+disease+recognize+alpha-synuclein+peptides.">T cells from patients with Parkinson's disease recognize alpha-synuclein peptides.</a> Nature. 546 (7660), 656-661 (2017).</li><li>Togo, T., 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=Occurrence+of+T+cells+in+the+brain+of+Alzheimer's+disease+and+other+neurological+diseases.">Occurrence of T cells in the brain of Alzheimer's disease and other neurological diseases.</a> Journal of Neuroimmunology. 124 (1), 83-92 (2002).</li><li>Parajuli, B., 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=Transnasal+transplantation+of+human+induced+pluripotent+stem+cell-derived+microglia+to+the+brain+of+immunocompetent+mice.">Transnasal transplantation of human induced pluripotent stem cell-derived microglia to the brain of immunocompetent mice.</a> Glia. 69 (10), 2332-2348 (2021).</li><li>Elmore, M. R., 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=Colony-stimulating+factor+1+receptor+signaling+is+necessary+for+microglia+viability,+unmasking+a+microglia+progenitor+cell+in+the+adult+brain.">Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain.</a> Neuron. 82 (2), 380-397 (2014).</li><li>Zasshi, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guiding+principles+for+the+care+and+use+of+animals+in+the+field+of+physiological+sciences.">Guiding principles for the care and use of animals in the field of physiological sciences.</a> Journal of the Physiologic Society of Japan. 64 (7-8), 143-146 (2002).</li><li>Bohlen, C. J., 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=Diverse+requirements+for+microglial+survival,+specification,+and+function+revealed+by+defined-medium+cultures.">Diverse requirements for microglial survival, specification, and function revealed by defined-medium cultures.</a> Neuron. 94 (4), 759-773 (2017).</li><li>Butovsky, O., 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=Identification+of+a+unique+TGF-beta-dependent+molecular+and+functional+signature+in+microglia.">Identification of a unique TGF-beta-dependent molecular and functional signature in microglia.</a> Nature Neuroscience. 17 (1), 131-143 (2014).</li><li>Regateiro, F. S., Howie, D., Cobbold, S. P., Waldmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+in+transplantation+tolerance.">TGF-beta in transplantation tolerance.</a> Current Opinion in Immunology. 23 (5), 660-669 (2011).</li><li>Yoshimura, A., Muto, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+function+in+immune+suppression.">TGF-beta function in immune suppression.</a> Current Topics in Microbiology and Immunology. 350, 127-147 (2011).</li></ol>

The authors have nothing to disclose.

The protocol here describes the non-invasive transplantation of iPSMG into the mouse brain. The uniqueness of the current protocol is that by combining pharmacological PLX ON/OFF methods and intranasal transplantation, iPSMG can be non-invasively transplanted into the immunocompetent mouse brain. Transplanted iPSMG formed the majority of microglia in the hippocampus and cerebellum by occupying the vacant niche for up to 60 days but not in the cortex.
The critical points for the efficient Tsn of iPSMG are (i) depletion efficiency of endogenous mouse microglia (ii) the administration of human cytokines every 12 h. Microglia maintain their own territory in the brain. Efficient depletion of mouse microglia is required to provide a niche for the engraftment of transplanted iPSMG. When depletion of endogenous mouse microglia is insufficient, colonization of the mouse hippocampus and cerebellum by iPSMG are not observed. The viability of microglia depends on CSF1R and TGFBR signaling19,21,22. hCSF1 is reported to selectively increase the viability of human microglia, and hTGF-&#946;1 is required for the viability of microglia as well as dampens inflammation when administered every 12 h21,23,24. In the absence of exogenous human cytokines, iPSMG are not observed in mouse brain. Further, care must be taken not to mechanically activate iPSMG by excessive pipetting or by any other means prior to Tsn, as it irrevocably alters iPSMG characteristics as well as transplantation efficiency. If the satisfactory Tsn of iPSMG is not seen, the viability of iPSMG before transplantation as well as the depletion of endogenous microglia, must be determined. If the depletion of endogenous mouse microglia is not more than 90%, PLX5622 feeding time may be modified to increase depletion.
Compared to a conventional surgical transplantation method that is invasive and requires additional equipment and training, Tsn allows transplantation in a non-invasive, simple, stable, and easy way.&#160;In addition, this method allows the transplantation of iPSMG into immunocompetent mice brains; thus, immunocompetent disease model mice can be used to study the response of iPSMG.
The greatest disadvantage of the current method is the regional heterogeneity in the engraftment of iPSMG. If the brain region-specific iPSMG transplantation is required, the current protocol is not suitable as the transplanted iPSMG remains engrafted for 60 days only in the hippocampus and the cerebellum but not in the cortex. Further, the need to administer exogenous human cytokines intranasally every 12 h is also a limitation of the current protocol as it requires extensive labor and is expensive.
In conclusion, a detailed protocol for Tsn of iPSMG into the brains of immunocompetent mice is provided. When combined with pharmacological ON/OFF of mouse microglia by PLX5622, this protocol allows successful engraftment of iPSMG. As transplanted cells can be observed in the hippocampus and cerebellum for a sustained period of time when exogenous cytokines are applied, the current method may be valuable for evaluating the role of human microglia in both physiological and pathological states in those regions.

Microglia are a specialized population of macrophage-like cells in the central nervous system (CNS) and play essential roles in controlling various brain functions like neural circuit development, modulating neurotransmission, and maintaining brain homeostasis1,2,3. Although murine microglia share many functions with ones from humans, they show species-specific differences. Thus, the response of mouse microglia to various stimuli may not always represent that of human microglia4,5,6. Although many studies have analyzed human microglia, those experiments are limited to in vitro studies. In vitro cultured human microglia show morphological features and gene expression that are very different from those in vivo. Thus, in vitro experiments may not always capitulate the in vivo characteristics of human microglia. Therefore, an experimental system to study human microglia in vivo is needed.
Recently, to study the in vivo characteristics of human microglia, in vitro generated induced pluripotent stem cells (iPSCs)- or embryonic stem cells-derived human microglia are surgically transplanted into mice brain7,8,9,10,11,12,13,14. Using this approach, various in vivo features of human microglia have been characterized. However, the widespread use of this method is limited for two reasons. First is the requirement of immune-deficient mice. Thus, to study the role of human microglia in various neurodegenerative diseases, disease mutation-carrying mice have to be crossed into immune-deficient mice, which requires significant time and effort. Furthermore, in various neurological disorders, peripheral immune cells, like T cells, can modulate microglial functions15,16,17. Therefore, experiments performed in immune-deficient mice may not represent bona fide characteristics of human microglia in vivo. Second, invasive surgeries to transplant microglia require additional equipment and training. Further, brain injury during invasive transplantation may change microglial phenotypes.
In this protocol, non-invasive transnasal transplantation (Tsn) of iPSMG into immunocompetent wild-type mice is described18. Combining pharmacological ON/OFF of a CSF1R antagonist PLX5622 which depletes endogenous mouse microglia19 and Tsn, iPSMG can be non-invasively transplanted into the mouse brain. Further, with the application of exogenous human cytokine, the transplanted iPSMG remains viable for 60 days in a region-specific manner without any immunosuppressants.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10566</td>
			<td></td>
		</tr>
		<tr>
			<td>AIN 93G</td>
			<td>Oriental Yeast Co</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Iba1 antibody</td>
			<td>FUJIFILM</td>
			<td>019&#8211;19741</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-STEM121 antibody</td>
			<td>Takara Bioscience</td>
			<td>Y40410</td>
			<td></td>
		</tr>
		<tr>
			<td>Butorphanol tartrate</td>
			<td>Kyoritsu Seiyaku</td>
			<td>8019</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus</td>
			<td>FV1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30070.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Frozen iPSMG</td>
			<td>Shionogi &#38; Co., Ltd</td>
			<td></td>
			<td>Laboratory for Drug Discovery and Disease Research</td>
		</tr>
		<tr>
			<td>Human colony stimulating factor 1 (hCSF1)</td>
			<td>PeproTech</td>
			<td>300-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Sigma-Aldrich</td>
			<td>H-3506</td>
			<td></td>
		</tr>
		<tr>
			<td>Medetomidine hydrochloride</td>
			<td>Meiji Seika</td>
			<td>VETLI5</td>
			<td></td>
		</tr>
		<tr>
			<td>Midazolam</td>
			<td>Astellas</td>
			<td>18005A2</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Wako Pure Chemical Industries</td>
			<td>162-16065</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>Eppendorf</td>
			<td>3120000011</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tip</td>
			<td>Eppendorf</td>
			<td>30076028</td>
			<td></td>
		</tr>
		<tr>
			<td>PLX5622</td>
			<td>Amadis Chemical</td>
			<td>A930097</td>
			<td></td>
		</tr>
		<tr>
			<td>Transforming growth factor-&#946;1 (Tgf-b1)</td>
			<td>PeproTech</td>
			<td>100-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X-100</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTA SHIELD Hard Set Mounting Medium</td>
			<td>Vector Laboratories</td>
			<td>H-1400-10</td>
			<td>antifade mounting medium</td>
		</tr>
	</tbody>
</table>

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.

This technique allows the investigator to non-invasively transplant iPSMG into the hippocampus and cerebellum but not into the cortex of the mouse brain. After completing the study, anesthetized mice were subjected to transcardial perfusion of ice-cold PBS(&#8722;), followed by ice-cold 4% (w/v) paraformaldehyde in PBS. The brains were isolated, postfixed overnight in 4% (w/v) paraformaldehyde, and cryoprotected in a PBS containing 30% (w/v) sucrose. Further, the brains were frozen in an embedding compound and sectioned (20 &#181;m thick coronal sections) on a cryostat. The sections were washed thrice in PBS(&#8722;) (10 min each) and permeabilized and blocked with 0.5% (v/v) Triton X-100 in 10% normal goat serum for 1 h. The sections were then incubated with anti-human-specific cytoplasm marker, STEM121 (1:100), and anti Iba1 (1:1000) for 5 days. Next, the sections were washed three times with PBS(&#8722;) for 10 min each and were incubated with secondary antibodies: Alexa Fluor 488-, or 546-conjugated mouse, or rabbit IgGs (1:1000) for 2 h at room temperature. After washing with PBS(-) three times, sections were mounted on slides using an antifade mounting medium. A confocal microscope equipped with a 40x objective lens was used for acquiring fluorescence images. The viability of iPSMG at 2 months post-transplantation in the hippocampus and the cortex are shown in Figure 1. The number of transplanted cells can be determined by counting cells that are positive for both human-specific antibodies and pan-microglial/monocyte markers, whereas endogenous mouse microglia are positive for pan-microglial/monocyte marker only as previously described18. The transplanted iPSMGs replace mouse microglia, show ramified morphologies in the hippocampus, and are not detected in the cortex18.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63574/63574fig01.jpg" /> 
Figure 1: Viability of iPSMG in the cortex and the hippocampus at 2 months after Tsn. The left panel shows immunostaining with a pan-microglial/monocyte marker, Iba1 (green). The middle panel shows immunostaining with human-specific cytoplasm marker STEM121 (red). The right panel shows a merged image of Iba1 and STEM121 immunostaining. (A) In control mice, only mouse microglia (Iba1+/STEM121-) were detected in both cortex and hippocampus. (B) In iPSMG transplanted mice, in the cortex, only mouse microglia (Iba1+/STEM121-) were detected, whereas in the hippocampus iPSMG (Iba1+/STEM121+) were detected. The arrows in the image show mouse microglia, whereas arrowheads show iPSMG. A confocal microscope equipped with a 40x objective lens was used for fluorescence image acquisition. Maximum image size: 1024 x 1024 pixels. Zoom factor: 2. Scale bars = 50 &#181;m <a href="https://www.jove.com/files/ftp_upload/63574/63574fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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 ...

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Research

JoVE Journal

Neuroscience

2.8K Views.  University of Yamanashi. 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.

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

Low-stress Route Learning Using the Lashley III Maze in Mice

Many behavior tests designed to assess learning and memory in rodents, particularly mice, rely on visual cues, food and/or water deprivation, or other aversive stimuli to motivate task acquisition. As animals age, sensory modalities deteriorate. For example, many strains of mice develop hearing deficits or cataracts. Changes in the sensory systems required to guide mice during task acquisition present potential confounds in interpreting learning changes in aging animals. Moreover, the use of aversive stimuli to motivate animals to learn tasks is potentially confounding when comparing mice with differential sensitivities to stress. To minimize these types of confounding effects, we have implemented a modified version of the Lashley III maze. This maze relies on route learning, whereby mice learn to navigate a maze via repeated exposure under low stress conditions, e.g. dark phase, no food/water deprivation, until they navigate a path from the start location to a pseudo-home cage with 0 or 1 error(s) on two consecutive trials. We classify this as a low-stress behavior test because it does not rely on aversive stimuli to encourage exploration of the maze and learning of the task. The apparatus consists of a modular start box, a 4-arm maze body, and a goal box. At the end of the goal box is a pseudo-home cage that contains bedding similar to that found in the animal&#x2019;s home cage and is specific to each animal for the duration of maze testing. It has been demonstrated previously that this pseudo-home cage provides sufficient reward to motivate mice to learn to navigate the maze1. Here, we present the visualization of the Lashley III maze procedure in the context of evaluating age-related differences in learning and memory in mice along with a comparison of learning behavior in two different background strains of mice. We hope that other investigators interested in evaluating the effects of aging or stress vulnerability in mice will consider this maze an attractive alternative to behavioral tests that involve more stressful learning tasks and/or visual cues.

The Lashley III maze is a route-learning task that does not rely on aversive stimuli or visual cues. It is thus a highly attractive option for evaluating learning and memory, especially in aging mice or otherwise where stress is a consideration.

Many behavior tests designed to assess learning and memory in rodents, particularly mice, rely on visual cues, food and/or water deprivation, or other ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions in newborn infants. While mosquitos are the main route of viral transmission, it has also been shown to spread via sexual contact and vertical mother-to-fetus transmission. In this latter case of transmission, due to the unique viral tropism of ZIKV, the virus is believed to predominantly target the neural progenitor cells (NPCs) of the developing brain.
Here a method for modeling ZIKV infection, and the resulting microcephaly, that occur when human cerebral organoids are exposed to live ZIKV is described. The organoids display high levels of virus within their neural progenitor population, and exhibit severe cell death and microcephaly over time. This three-dimensional cerebral organoid model allows researchers to conduct species-matched experiments to observe and potentially intervene with ZIKV infection of the developing human brain. The model provides improved relevance over standard two-dimensional methods, and contains human-specific cellular architecture and protein expression that are not possible in animal models.

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

This protocol describes a technique used to model Zika virus infection of the developing human brain. Using wildtype or engineered stem cell lines, researchers may use this technique to uncover the various mechanisms or treatments that may affect early brain infection and resulting microcephaly in Zika virus-infected embryos.

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven therapies for TBI have been developed. This paper presents a novel method for pre-clinical neurotrauma research intended to complement existing pre-clinical models. It introduces human pathophysiology through the use of human induced pluripotent stem cell-derived neurons (hiPSCNs). It achieves loading pulse duration similar to the loading durations of clinical closed head impact injury. It employs a 96-well format that facilitates high throughput experiments and makes efficient use of expensive cells and culture reagents. Silicone membranes are first treated to remove neurotoxic uncured polymer and then bonded to commercial 96-well plate bodies to create stretchable 96-well plates. A custom-built device is used to indent some or all of the well bottoms from beneath, inducing equibiaxial mechanical strain that mechanically injures cells in culture in the wells. The relationship between indentation depth and mechanical strain is determined empirically using high speed videography of well bottoms during indentation. Cells, including hiPSCNs, can be cultured on these silicone membranes using modified versions of conventional cell culture protocols. Fluorescent microscopic images of cell cultures are acquired and analyzed after injury in a semi-automated fashion to quantify the level of injury in each well. The model presented is optimized for hiPSCNs but could in theory be applied to other cell types.

Here we present a method for a human in vitro model of stretch injury in a 96-well format on a timescale relevant to impact trauma. This includes methods for fabricating stretchable plates, quantifying the mechanical insult, culturing and injuring cells, imaging, and high content analysis to quantify injury.

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven ...

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have identified a special population of vascular cells, the periventricular endothelial cells, that play a critical role in the migration and distribution of forebrain GABAergic interneurons during embryonic development. This, coupled with their cell-autonomous functions, alludes to novel roles of periventricular endothelial cells in the pathology of neuropsychiatric disorders like schizophrenia, epilepsy, and autism. Here, we have described three different in vitro assays that collectively evaluate the functions of periventricular endothelial cells and their interaction with GABAergic interneurons. Use of these assays, particularly in a human context, will allow us to identify the link between periventricular endothelial cells and brain disorders. These assays are simple, low cost, and reproducible, and can be easily adapted to any adherent cell type.

We present three simple in vitro assays-the long-distance migration assay, the co-culture migration assay, and chemo-attraction assay-that collectively evaluate the functions of human stem cell derived periventricular endothelial cells and their interaction with GABAergic interneurons.

Role of brain vasculature in nervous system development and etiology of brain disorders is increasingly gaining attention. Our recent studies have ...

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human neurons, which may contribute to neurodegeneration in various disease states. Although it is challenging to examine mitochondrial dynamics in live human nerves, such paradigms are critical for studying the role of mitochondria in neurodegeneration. Described here is a protocol for analyzing mitochondrial transport and mitochondrial morphology in forebrain neuron axons derived from human induced pluripotent stem cells (iPSCs). The iPSCs are differentiated into telencephalic glutamatergic neurons using well-established methods. Mitochondria of the neurons are stained with MitoTracker CMXRos, and mitochondrial movement within the axons are captured using a live-cell imaging microscope equipped with an incubator for cell culture. Time-lapse images are analyzed using software with &#34;MultiKymograph&#34;, &#34;Bioformat importer&#34;, and &#34;Macros&#34; plugins. Kymographs of mitochondrial transport are generated, and average mitochondrial velocity in the anterograde and retrograde directions is read from the kymograph. Regarding mitochondrial morphology analysis, mitochondrial length, area, and aspect ratio are obtained using the ImageJ. In summary, this protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology to facilitate studies of neurodegenerative diseases.

Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases. The presented protocol uses induced pluripotent stem cell-derived forebrain neurons to assess mitochondrial transport and morphology in hereditary spastic paraplegia. This protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology, which will facilitate the study of neurodegenerative disease.

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human ...

Obtaining Human Microglia from Adult Human Brain Tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining homeostasis, and during infection or injury. Several independent research groups have highlighted the central role that microglia play in autoimmune diseases, autoinflammatory syndromes and cancers. The activation of microglia in some neurological diseases may directly participate in pathogenic processes. Primary microglia are a powerful tool to understand the immune responses in the brain, cell-cell interactions and dysregulated microglia phenotypes in disease. Primary microglia mimic in vivo microglial properties better than immortalized microglial cell lines. Human adult microglia exhibit distinct properties as compared to human fetal and rodent microglia. This protocol provides an efficient method for isolation of primary microglia from adult human brain. Studying these microglia can provide critical insights into cell-cell interactions between microglia and other resident cellular populations in the CNS including, oligodendrocytes, neurons and astrocytes. Additionally, microglia from different human brains may be cultured for characterization of unique immune responses for personalized medicine and a myriad of therapeutic applications.

This protocol is an efficient, cost effective and robust method of isolating primary microglia from live, adult, human brain tissue. Isolated primary human microglia can serve as a tool for studying cellular processes in homeostasis and disease.

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining ...

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.

Presented here is a surgical technique for transplanting human pluripotent stem cell (hPSC)-derived retinal tissue into the subretinal space of a large animal model.

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and ...

Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential (MMP), mitochondrial production of reactive oxygen species (ROS), and mitochondrial DNA (mtDNA) copy number are often features of these processes. This report details a novel flow cytometry-based approach to measure multiple mitochondrial parameters in different cell types, including human induced pluripotent stem cells (iPSCs) and iPSC-derived neural and glial cells. This flow-based strategy uses live cells to measure mitochondrial volume, MMP, and ROS levels, as well as fixed cells to estimate components of the mitochondrial respiratory chain (MRC) and mtDNA-associated proteins such as mitochondrial transcription factor A (TFAM). 
By co-staining with fluorescent reporters, including MitoTracker Green (MTG), tetramethylrhodamine ethyl ester (TMRE), and MitoSox Red, changes in mitochondrial volume, MMP, and mitochondrial ROS can be quantified and related to mitochondrial content. Double staining with antibodies against MRC complex subunits and translocase of outer mitochondrial membrane 20 (TOMM20) permits the assessment of MRC subunit expression. As the amount of TFAM is proportional to mtDNA copy number, the measurement of TFAM per TOMM20 gives an indirect measurement of mtDNA per mitochondrial volume. The entire protocol can be carried out within 2-3 h. Importantly, these protocols allow the measurement of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry.

This study reports a novel approach to measure multiple mitochondrial functional parameters based on flow cytometry and double staining with two fluorescent reporters or antibodies to detect changes in mitochondrial volume, mitochondrial membrane potential, reactive oxygen species level, mitochondrial respiratory chain composition, and mitochondrial DNA.

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential ...