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>

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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><tbody><tr><td>Dulbecco&#039;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&amp;ndash;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 &amp;amp; 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-&amp;beta;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>

Watch this Scientific Journal Video about Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route at JoVE.com

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 ...

transplantation-human-induced-pluripotent-stem-cell-derived-microglia

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2.8K Views.  University of Yamanashi. Dit protocol maakt een niet-invasieve transplantatie van iPSMG in immunocompetente muizenhersenen mogelijk door farmacologische aan/uit van een CSF1-receptorantagonist PLX5622 te combineren met transnasale transplantatie. Het belangrijkste voordeel van deze techniek is dat iPSMG kan worden getransplanteerd zonder hersenschade te veroorzaken, zelfs bij immunocompetente muizen. Microgliale transplantatie kan dienen als een potentiële therapie om disfunctionele microglia uit te wisselen met fun...

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

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 ...

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high ...

Medicine

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

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

Immunology and Infection

Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of therapeutic stem cells via the intranasal route is a recently discovered alternative strategy, with strong potential for clinical translation, due to its non-invasive nature compared to intracranial implantation or delivery via systemic routes. The lack of blood brain barrier further strengthens the therapeutic potential of stem cells undergoing intranasal brain entry. This article summarizes the essential techniques utilized in our studies and outlines the basic principles of intranasal strategy for stem cell delivery using a mouse model of intracranial glioma xenografts. We demonstrate the optimized procedures that generate consistent and reproducible results with specific predetermined experimental parameters and offer guidelines for streamlined work flow that ensure efficient execution and reliable experimental outcome. The article is designed to serve as a baseline for further experimental customization based on hypothesis, stem cell types, or tumor specifics.

Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma.

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of ...

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 ...

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 ...

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point measurements and can therefore only evaluate longitudinal changes of cell migration and survival to a limited extent. This paper provides a reliable, minimally invasive protocol to transplant and longitudinally track neural progenitor cells (NPCs) in the adult mouse brain. Before transplantation, cells are transduced with a lentiviral vector comprising a bioluminescent (firefly-luciferase) and fluorescent (green fluorescent protein [GFP]) reporter. The NPCs are transplanted into the right cortical hemisphere using stereotaxic injections in the sensorimotor cortex. Following transplantation, grafted cells were detected through the intact skull for up to five weeks (at days 0, 3, 14, 21, 35) with a resolution limit of 6,000 cells using in vivo bioluminescence imaging. Subsequently, the transplanted cells are identified in histological brain sections and further characterized with immunofluorescence. Thus, this protocol provides a valuable tool to transplant, track, quantify, and characterize cells in the mouse brain.

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

We describe the intraparenchymal transplantation of human neural progenitor cells transduced with a dual reporter vector expressing luciferase-green fluorescent protein (GFP) in the mouse brain. After transplantation, the luciferase signal is repeatedly measured using in vivo bioluminescence and GFP-expressing grafted cells identified in brain sections using fluorescence microscopy.

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point ...

Developing Blood-Induced Microglia-Like Cells for Studying Human Brain Disorders

Engineering of Human Blood-Induced Microglia-like Cells for Reverse-Translational Brain Research

Recent investigations employing animal models have highlighted the significance of microglia as crucial immunological modulators in various neuropsychiatric and physical diseases. Postmortem brain analysis and positron emission tomography imaging are representative research methods that evaluate microglial activation in human patients; the findings have revealed the activation of microglia in the brains of patients presenting with various neuropsychiatric disorders and chronic pain. Nonetheless, the aforementioned technique merely facilitates the assessment of limited aspects of microglial activation. 
In lieu of brain biopsy and the induced pluripotent stem cell technique, we initially devised a technique to generate directly induced microglia-like (iMG) cells from freshly derived human peripheral blood monocytes by supplementing them with granulocyte-macrophage colony-stimulating factor and interleukin 34 for 2 weeks. These iMG cells can be employed to perform dynamic morphological and molecular-level analyses concerning phagocytic capacity and cytokine releases following cellular-level stress stimulation. Recently, comprehensive transcriptome analysis has been used to verify the similarity between human iMG cells and brain primary microglia. 
The patient-derived iMG cells may serve as key surrogate markers for predicting microglial activation in human brains and have aided in the unveiling of previously unknown dynamic pathophysiology of microglia in patients with Nasu-Hakola disease, fibromyalgia, bipolar disorder, and Moyamoya disease. Therefore, the iMG-based technique serves as a valuable reverse-translational tool and provides novel insights into elucidating dynamic the molecular pathophysiology of microglia in a variety of mental and physical diseases.

Author Spotlight: Induced Microglia-Like Cell Technology to Shed Light on the Role of Microglial Dysfunction in Neuropsychiatric Disorders

This study delineates a novel approach for the establishment of human monocyte-derived microglia-like (iMG) cells that enable the indirect assessment of brain inflammation. This presents a cellular model that may be beneficial to research focusing on potential inflammation of the brain and associated neuropsychiatric disorders.

Recent investigations employing animal models have highlighted the significance of microglia as crucial immunological modulators in various ...

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.

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.

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

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