Name: stem-cell based engineered immunity against hiv infection in the humanized mouse model
Uploaded: 2016-07-02T13:00:00.000+00:00
Duration: 11 min 38 s
Description: Watch this Scientific Journal Video about Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model at JoVE.com

We would like to thank Ms. Jessica Selander in providing artistic assistant in making our figures. This work was funded by grants from the NIAID/NIH, grant no. RO1AI078806, the UCLA Center for AIDS Research (CFAR), grant no. P30AI28697, the California Institute for Regenerative Medicine, grant no. TR4-06845, the American Federation for AIDS Research (amfAR), grant no. #108929-54-RGRL, and the UC Multi-campus Research Program and Initiatives, California Center for Antiviral Drug discovery (CCADD)

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The authors have nothing to disclose.

Con CAR y ganando impulso inmunidad ingeniería basada en HSC hacia los estudios clínicos, es importante tener un modelo animal adecuado para examinar de cerca la diferenciación y función de estas células modificadas. En este protocolo se describen los métodos para la construcción y prueba de ratones humanizados con células madre modificadas genéticamente manipulados contra el VIH. Es importante tener la transducción eficiente de células madre antes del trasplante. Sin embargo, debido a la capacidad de las células T para proliferar a partir del reconocimiento de las células diana, los bajos niveles de modificación de células madre eran suficientes para generar una respuesta sólida contra la replicación del VIH 8. No obstante, para lograr un alto nivel de modificación de células madre, se recomienda utilizar células CD34 + transducidas células en la mezcla de proteína gelatinosa con timo autólogo en lugar de trozos de hígado y timo para la cirugía de ratones que habían sido descritos en otras partes 13-15. mezcla de proteína gelatinosa es un solubilized tejido membrana basal rica en proteínas de la matriz extracelular. En condiciones fisiológicas normales, la mezcla de proteína gelatinosa polimeriza para producir una matriz reconstituida, biológicamente activa y estable que permitir la unión y la diferenciación efectiva de la célula madre 20. la reconstitución de células humanas se puede comprobar mediante sangrado retro-orbital y citometría de flujo 6 - 8 semanas después de la cirugía. Para asegurar que el vector no es tóxico para la supervivencia de células madre y la renovación, se recomienda para titular el vector en las células CD34 + antes del experimento como se describe en 1.2.6. La capacidad del modelo de ratón NSG-BLT para apoyar la infección de la mucosa, viremia consistente y respuestas inmunes celulares hace que sea un modelo muy útil para estudiar la patología inmune VIH y terapias basadas en células para el tratamiento de la infección por VIH 1. Lo más importante, las células T generadas a partir de los ratones NSG-BLT se seleccionan en el tejido tímico autólogo, permitiendo que el investigador para estudiar el destinode modificación de genes de células madre después de la selección tímica 8,21. Con el método descrito, hemos sido capaces de conseguir un 40% -90% de la reconstitución de las células inmunes humanas constantemente. Los bajos niveles de reconstitución de células humanas pueden resultar de múltiples factores, incluyendo las capacidades de la persona que realiza la cirugía y la calidad de los tejidos para el trasplante. Para lograr un alto nivel de reconstitución de células humanas, es importante para asegurar que los trasplantes se colocan de forma segura debajo de la cápsula renal. Además, es muy recomendable para examinar cada implante del timo preparada para la cirugía bajo microscopio de luz y desechar piezas cuestionables. Aunque el modelo de BLT ratón humanizado es una herramienta prometedora para el estudio de la inmunidad contra el VIH ingeniería (revisado en 1,15,22), tiene sus propias limitaciones. A saber, este modelo no imita perfectamente un sistema inmune periférica humana. Los estudios han demostrado el desarrollo de problemas de hiper-mutado, Antibo IgG cambio de clasedy 1,23. Además, el uso de ratones inmunodeficientes es un desafío técnico y el mantenimiento de estos ratones sanos requiere considerables recursos y formación. infecciones oportunistas sutiles diferencias pueden manifestarse como significativas entre las muestras y potencialmente tener consecuencias negativas en la experimentación. Por lo tanto, es importante contar con instalaciones bien preparados y personal debidamente capacitado para mantener la integridad de la futura 1,24 datos. Con estas limitaciones en mente, el modelo de ratón humanizado GSN-BLT todavía proporciona una herramienta importante para el estudio de la inmunidad de ingeniería basada en células madre, como lo demuestran estos ejemplos 4,8. Con la tendencia de desarrollar receptores de antígenos quiméricos basado en una amplia VIH anticuerpos neutralizantes 10 y modificación del dominio de señalización para CAR más eficiente 25, este modelo y el protocolo se pueden utilizar para caracterizar e investigar la funcionalidad del gen modificado cells con una nueva generación de automóviles. Además, este modelo puede potencialmente dar cabida a los estudios sobre la terapia basada inmune (tales como el bloqueo del receptor inhibidor) en conjunción con la inmunidad de ingeniería.

A pesar del éxito de la terapia anti-retroviral combinada, la infección por VIH sigue siendo una enfermedad de por vida. La respuesta inmune celular contra el VIH juega muy importante papel en el control de la replicación del VIH. Los recientes avances en la manipulación de células madre ha permitido para el rápido desarrollo de la terapia génica enfoques para el tratamiento del VIH 1-3. Como resultado de ello, es importante tener un modelo animal adecuado que permite el estudio in vivo de la eficacia de las terapias basadas en células contra el VIH. Trabajando con el VIH en modelos animales se complica por el hecho de que el virus sólo infecta las células humanas. Para evitar esta limitación, los científicos han recurrido al uso de modelos de enfermedades como el virus de inmunodeficiencia de los simios (SIV) en macacos Rhesus 4,5. Por desgracia, existen importantes limitaciones en este modelo debido a las diferencias inherentes entre las especies y las diferencias entre SIV y el VIH. Además, sólo las instalaciones altamente especializados son capable de apoyar el trabajo con los primates no humanos y macacos cada uno requiere una gran inversión. Por lo tanto, hay una necesidad urgente de un modelo que utiliza el sistema inmunitario humano, que es susceptible a la infección por VIH / patogénesis, y es menos económicamente prohibitivo. El diabético no obeso (NOD) -severe inmunodeficiencia combinada (SCID) de la cadena gamma nocaut -común (c γ - / -) (o NSG) Sangre / Hígado / timo (BLT) modelo de ratón humanizado se demuestra cada vez más como una herramienta importante para estudiar la infección por el VIH. Mediante la implantación de células madre hematopoyéticas (HSC) y timo fetal, los ratones son capaces de desarrollar y recapitular un sistema inmunológico humano 1-3. Un tipo de terapia génica basada en células madre implica &#39;redirigir &quot;células T periféricas a diana del VIH mediante la reprogramación de las células madre hematopoyéticas (HSC) para diferenciarse en células T específicas de antígeno. Hemos demostrado previamente que las CMH de ingeniería con un anti-VIH específicos de células T re clonado molecularCeptor (TCR) contra el epítopo SL9 (aminoácido 77 a 85; SLYNTVATL) del VIH-1 Gag puede redirigir células madre en células formadoras de T maduras que suprimen la replicación del VIH en el GSN-BLT modelo de ratón humanizado 6. La advertencia de utilizar un TCR clonado molecular es que se limita a un subtipo específico del antígeno leucocitario humano (HLA) que limitar la aplicación de esta terapia. receptores de antígenos quiméricos (CAR), por el contrario, pueden aplicarse universalmente a todos los subtipos de HLA. Se llevaron a cabo estudios iniciales utilizando un CAR construido con los dominios extracelulares y transmembrana de CD4 humano fusionado al dominio intracelular de señalización CD3 ζ (denominado el CD4ζCAR). CD4ζCAR expresa en las células CD8 T pueden reconocer envoltura del VIH y desencadenar una respuesta de células T citotóxicas que es similar a la mediada por un receptor de células T 7. Recientemente hemos demostrado que HSCs humanas se pueden modificar con CD4ζCAR, que puede diferenciarse en múltiples li hematopoyéticoneages, incluyendo las células T funcionales capaces de suprimir la replicación del VIH en el modelo de ratón humanizado 8. Con el rápido avance en las terapias de los receptores de antígeno quiméricos para el cáncer de 9, y la caracterización en curso de anticuerpos neutralizantes amplias potentes 10-12 contra el VIH que permiten la construcción de las CAR de anticuerpos de cadena única, es perceptible que muchos nuevos constructos candidatos, además de CD4ζCAR , será generado y probado para la terapia génica basada en células madre de enfermedades de VIH y otras enfermedades. Además, el modelo de ratón humanizado GSN-BLT que contiene estos coches específicos de antígeno también puede proporcionar una herramienta útil para examinar de cerca las respuestas de células T humanas in vivo. Es importante destacar que nuestro protocolo difiere de los métodos anteriores descritos para la construcción de humanizada de ratones BLT 13-15 en que el HSCs en mezcla de proteína gelatinosa se ​​utiliza en lugar de los troncos de hígado fetal 16. Este protocolo describe: 1) la construcción de humanilos ratones BLT zed ingeniería con CD4ζCAR; y 2) la caracterización de la diferenciación de las células modificadas genéticamente; y 3) la caracterización de la funcionalidad de las células modificadas genéticamente.

<table><tbody><tr><td>CD34 microbead kit</td><td>Miltenyi</td><td>130-046-702</td><td>For sorting human CD34+ progenitor cells</td></tr><tr><td>Bambanker</td><td>Wako</td><td>302-14681</td><td>For freezing cells</td></tr><tr><td>QIAamp Viral RNA kit&amp;nbsp;</td><td>Qiagen</td><td>52904</td><td>For measuring viral load in the serum</td></tr><tr><td>MACSQuant Flow Cytometer</td><td>Miltenyi</td><td></td><td>For flow analysis</td></tr><tr><td>BD LSRFortessa&amp;trade;</td><td>BD biosciences</td><td></td><td>For flow analysis</td></tr><tr><td>Hyaluronidase</td><td>Sigma</td><td>H6254-500MG&amp;nbsp;</td><td>For tissue digestion</td></tr><tr><td>Deoxyribonuclease I&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Worthington</td><td>LS002006&amp;nbsp;</td><td>for tissue digestion</td></tr><tr><td>Collagenase</td><td>Life technology</td><td>17104-019&amp;nbsp;</td><td>for tissue digestion</td></tr><tr><td>CFX Real time PCR detection system</td><td>Biorad</td><td></td><td>For measuring viral load and gene expression</td></tr><tr><td>Mice, strain NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ</td><td>The Jackson Laboratory</td><td>5557</td><td>For constructing the humanized mice</td></tr><tr><td>Penicillin Streptomycin (Pen Strep)</td><td>Thermo Fisher Scientific</td><td>10378016</td><td>For culturing cells</td></tr><tr><td>Piperacillin/tazobactam</td><td>Pfizer</td><td>Zosyn</td><td>Anti-fungal</td></tr><tr><td>Amphotericin B (Fungizone antimycotic)</td><td>Thermo Fisher Scientific</td><td>15290-018</td><td>Anti-fungal</td></tr><tr><td>Autoclip Wound Clips, 9 mm - 1,000 units</td><td>Becton Dickinson</td><td>427631&amp;nbsp;</td><td>For surgery</td></tr><tr><td>Sterile Poly-Reinforced Aurora Surgical Gowns, 30 per case&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Medline</td><td>DYNJP2707&amp;nbsp;</td><td>For surgery</td></tr><tr><td>Sutures, 4-0, vicryl&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Owens and Minor</td><td>23000J304H&amp;nbsp;&amp;nbsp;</td><td>For surgery</td></tr><tr><td>Alcohol prep pads&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Owens and Minor</td><td>3583006818</td><td>For surgery</td></tr><tr><td>Gloves, surgical, 6 1/2</td><td>Owens and Minor</td><td>4075711102</td><td>For surgery</td></tr><tr><td>Yssel&amp;rsquo;s Serum-Free T-Cell Medium</td><td>Gemini Bio-products</td><td>400-102</td><td>For CD34+ cell transduction</td></tr><tr><td>Human Serum Albumin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A9511</td><td>For CD34+ cell transduction</td></tr></tbody></table>

stem-cell based engineered immunity against hiv infection in the humanized mouse model

Con el rápido desarrollo de terapias génicas basadas en células madre contra el VIH, no hay presión requisito de un modelo animal para estudiar la diferenciación hematopoyética y la función inmune de las células modificadas genéticamente. El humanizado de médula ósea / del hígado / timo (BLT) modelo de ratón permite la completa reconstitución de un sistema inmunológico humano en la periferia, que incluye las células T, células B, células NK y monocitos. El implante del timo humano también permite la selección tímica de las células T en el tejido tímico autólogo. Además del estudio de la infección por VIH, el modelo se presenta como una herramienta poderosa para estudiar la diferenciación, el desarrollo y la funcionalidad de las células derivadas de células madre hematopoyéticas (HSC). Aquí describimos la construcción de humanizado diabéticos no obesos (NOD) -severe inmunodeficiencia combinada (SCID) de la cadena gamma nocaut -común (c γ - / -) ratones -Bone ósea / hígado / timo (NSG-BLT) con HSC transducidas con receptor de antígeno quimérico CD4 (CD4CAR)vector lentivirus. Se demuestra que la CD4CAR HSC puede diferenciar con éxito en múltiples linajes y tienen actividad anti-VIH. El objetivo del estudio es demostrar el uso de modelo de ratón NSG-BLT como un modelo in vivo para la inmunidad contra el VIH ingeniería. Vale la pena señalar que, dado que se utiliza lentivirus y el tejido humano, los experimentos y las cirugías deben realizarse en una cabina de bioseguridad de clase II en un nivel de bioseguridad 2 (BSL2) con precauciones especiales instalación (BSL2 +).

La Figura 1 muestra un esquema de la construcción de ratones humanizados BLT con células madre modificada. 10 semanas después de la cirugía de implante, se sacrificaron los ratones para evaluar la diferenciación y el desarrollo de células modificadas de genes. Como se muestra en la Figura 2, múltiples tejidos linfoides (sangre, bazo, timo y médula ósea) se recogieron de un ratón que se ha modificado con CD4ζCAR. El CD4ζCAR utilizado en este protocolo contiene CD4 receptor de antígeno quimérico y GFP que puede ser detectada por el anticuerpo anti-CD4 y la expresión de GFP 8. Se aislaron las células y se tiñeron con anticuerpos contra CD45 humano, así como de anticuerpos anti-CD4 y se analizaron por citometría de flujo. se detectaron GFP y células positivas dobles CD4, lo que indica la presencia de células CD4CAR + en múltiples tejidos linfoides. Para investigar la diferenciación de las células modificadas de genes,<html> los esplenocitos se tiñeron con anticuerpos contra CD45 humano (linfocitos), CD3 (células T), CD19 (células B), CD14 (monocitos y macrófagos) y CD337 (células NK). Como se muestra en la Figura 3, las células madre hematopoyéticas CD4ζCAR diferenciarse en múltiples linajes. Para investigar si las células CD4CAR modificados son funcionales, que coincubated esplenocitos con células diana que las células CD4CAR reconocerían (T1 células infectadas por el VIH o las células no infectadas T1 como control). Las células se co-incubaron durante la noche y se añadió el inhibidor del transporte de la proteína durante 6 horas adicionales. Después, las células se fijaron y se permeabilizaron para la tinción para la expresión intracelular de citoquinas tales como IFN y TNF. Como se muestra en la Figura 4, las células que expresan CAR producen mayor cantidad de IFN y TNF con células T1 infectados. carga / 54048 / 54048fig1.jpg &quot;/&gt; Figura 1: Esquema de construcción de los ratones humanizados BLT con células madre modificadas FT:. Timo fetal. FL:. Hígado fetal <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54048/54048fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/54048/54048fig2.jpg" /> Figura 2:. Células modificadas CD4 receptor de antígeno quimérico se pueden detectar en varios tejidos linfoides de ratón con HSCs modificado CD4CAR se sacrificaron 10 semanas después de la cirugía y múltiples tejidos linfoides fueron cosechadas y las células se tiñeron con anticuerpos CD4 CD45 anti-humano y anti-humanos y se analizaron mediante citometría de flujo. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54048/54048fig2large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> Figura 3:.. Células modificadas CD4 de los receptores de antígeno quimérico pueden diferenciarse en múltiples linajes esplenocitos de ratones modificados CD4CAR se cosecharon y se tiñeron con anticuerpos contra CD45 humano, CD3, CD19, CD14 y CD337 y se analizó mediante citometría de flujo <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54048/54048fig3large.jpg" target="_blank">favor, haga clic aquí para ver una mayor versión de esta figura.</a> <img alt="Figura 4" src="/files/ftp_upload/54048/54048fig4.jpg" /> Figura 4: Ej. Ensayo de citoquinas in vivo de las células CD4 + T CAR esplenocitos de los ratones infectados con el VIH CD4CAR se estimularon con células T1 infectado o no infectado por el VIH y su producción intracelular de citoquinas se muestra. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54048/54048fig4large.jpg" target="_blank">Por favor, haga clic aquí para competir</a>wa versión más grande de esta figura.

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Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

-Base de células madre Inmunidad Diseñado frente a la infección por VIH en el modelo de ratón humanizado

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

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10.1K vistas.  University of California, Los Angeles. This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection. 

Video: Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

Uso del ratón humanizado BLT como un modelo de células madre del tumor Terapia génica basada en

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

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Inmunología e Infección

Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine cancer models are extremely limited in their diversity and suffer from poor translation to the clinic. To serve as a more physiological preclinical model for immunotherapy studies, this protocol has been developed to evaluate the treatment of human tumors in a mouse reconstituted with a human immune system. This unique protocol demonstrates the development of human immune system (HIS, &#34;humanized&#34;) mice, followed by implantation of a human tumor, either a cell-line derived xenograft (CDX) or a patient derived xenograft (PDX). HIS mice are generated by injecting CD34+ human hematopoietic stem cells isolated from umbilical cord blood into neonatal BRGS (BALB/c Rag2-/- IL2R&#947;C-/- NODSIRP&#945;) highly immunodeficient mice that are also capable of accepting a xenogeneic tumor. The importance of the kinetics and characteristics of the human immune system development and tumor implantation is emphasized. Finally, an in-depth evaluation of the tumor microenvironment using flow cytometry is described. In numerous studies using this protocol, it was found that the tumor microenvironment of individual tumors is recapitulated in HIS-PDX mice; &#34;hot&#34; tumors exhibit large immune infiltration while &#34;cold&#34; tumors do not. This model serves as a testing ground for combination immunotherapies for a wide range of human tumors and represents an important tool in the quest for personalized medicine.

This protocol outlines the generation of human immune system (HIS) mice for immuno-oncology studies. Instructions and considerations in the use of this model for testing human immunotherapeutics on human tumors implanted in this model are presented with an emphasis on characterizing the response of the human immune system to the tumor.

Prueba de inmunoterapia contra el cáncer en un modelo de ratón humanizado con tumores humanos

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine ...

Investigación sobre el cáncer

Humanized NOD/SCID/IL2r&#947;null (hu-NSG) Mouse Model for HIV Replication and Latency Studies

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in high demand. With a close genetic and behavioral resemblance to humans, small animals such as the mouse are good candidates for human disease models, through which human-like symptoms and responses can be recapitulated. Further, the mouse genetic background can be altered to accommodate diverse demands. The NOD/SCID/IL2r&#947;null (NSG) mouse is one of the most widely used immunocompromised mouse strains; it allows engraftment with human hematopoietic stem cells and/or human tissues and the subsequent development of a functional human immune system. This is a critical milestone in understanding the prognosis and pathophysiology of human-specific diseases such as HIV/AIDS and aiding the search for a cure. Herein, we report a detailed protocol for generating a humanized NSG mouse model (hu-NSG) by hematopoietic stem cell transplantation into a radiation-conditioned neonatal NSG mouse. The hu-NSG mouse model shows multi-lineage development of transplanted human stem cells and susceptibility to HIV-1 viral infection. It also recapitulates key biological characteristics in response to combinatorial antiretroviral therapy (cART).

This protocol provides a method to establish humanized mice (hu-NSG) via intrahepatic injection of human hematopoietic stem cells into radiation-conditioned neonatal NSG mice. The hu-NSG mouse is susceptible to HIV infection and combinatorial antiretroviral therapy (cART) and serves as a suitable pathophysiological model for HIV replication and latency investigations.

(Hu-NSG) ratón humanizado del NOD/SCID/IL2rγnull modelo para estudios de latencia y replicación del VIH

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in ...

Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of their morbidity. The liver immunopathology caused by HIV-1 remains elusive. Small xenograft animal models with human hepatocytes and human immune system can recapitulate the human biology of the disease&#39;s pathogenesis. Herein, a protocol is described to establish a dual humanized mouse model through human hepatocytes and CD34+ hematopoietic stem/progenitor cells (HSPCs) transplantation, to study liver immunopathology as observed in HIV-infected patients. To achieve dual reconstitution, male TK-NOG (NOD.Cg-Prkdcscid Il2rgtm1Sug Tg(Alb-TK)7-2/ShiJic) mice are intraperitoneally injected with ganciclovir (GCV) doses to eliminate mouse transgenic liver cells, and with treosulfan for nonmyeloablative conditioning, both of which facilitate human hepatocyte (HEP) engraftment and human immune system (HIS) development. Human albumin (ALB) levels are evaluated for liver engraftment, and the presence of human immune cells in blood detected by flow cytometry confirms the establishment of human immune system. The model developed using the protocol described here resembles multiple components of liver damage from HIV-1 infection. Its establishment could prove to be essential for studies of hepatitis virus co-infection and for the evaluation of antiviral and antiretroviral drugs.

This protocol provides a reliable method to establish humanized mice with both human immune system and liver cells. Dual reconstituted immunodeficient mice achieved via intrasplenic injection of human hepatocytes and CD34+ hematopoietic stem cells are susceptible to human immunodeficiency virus-1 infection and recapitulate liver damage as observed in HIV-infected patients.

Establecimiento del modelo de ratón TK-NOG humanizado dual para la patogénesis hepática asociada al VIH

Despite the increased life expectancy of patients infected with human immunodeficiency virus-1 (HIV-1), liver disease has emerged as a common cause of ...

Chronic, Acute, and Reactivated HIV Infection in Humanized Immunodeficient Mouse Models

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical research on infectious diseases. Described here are three models of humanized immunodeficient mice for studying the dynamics of HIV infection. The first is based on the intrahepatic injection of CD34+ hematopoietic stem cells in newborn mice, which allows for the reconstitution of several blood and lymphoid tissue-confined cells, followed by infection with a reference HIV strain. This model allows monitoring for up to 36 weeks post-infection and is hence called the chronic model. The second and third models are referred to as the acute and reactivation models, in which peripheral blood mononuclear cells are intraperitoneally injected in adult mice. In the acute model, cells from a healthy donor are engrafted through the intraperitoneal route, followed by infection with a reference HIV strain. Finally, in the reactivation model, cells from an HIV-infected donor under antiretroviral therapy are engrafted via the intraperitoneal route. In this case, a drug-free environment in the mouse allows for virus reactivation and an increase in viral load. The protocols provided here describe the conventional experimental approach for humanized, immunodeficient mouse models of HIV infection.

Described here are three experimental approaches for studying the dynamics of HIV infection in humanized mice. The first permits the study of chronic infection events, whereas the two latter allows for the study of acute events after primary infection or viral reactivation.

Infección crónica, aguda y reactivada por VIH en modelos de ratón inmunodeficientes humanizados

Humanized NOD/SCID/IL-2 receptor &#947;-chainnull mice recapitulate some features of human immunity, which can be exploited in basic and pre-clinical ...

Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes the establishment of a human immune system in adult NOG mice. Here, we explain all the practical steps from isolation of umbilical cord blood derived human CD34+ cells and their subsequent intravenous transplantation into the mice, to the manipulation of the model through HIV infection, combination antiretroviral therapy (cART), and blood sampling. Approximately 75,000 hCD34+ cells are injected intravenously into the mice and the level of human chimerism, also known as humanization, in the peripheral blood is estimated longitudinally for months by flow cytometry. A total of 75,000 hCD34+ cells yields 20%&#8211;50% human CD45+ cells in the peripheral blood. The mice are susceptible to intravaginal infection with HIV and blood can be sampled once weekly for analysis, and twice monthly for extended periods. This protocol describes an assay for quantification of plasma viral load using droplet digital PCR (ddPCR). We show how the mice can be effectively treated with a standard-of-care cART regimen in the diet. The delivery of cART in the form of regular mouse chow is a significant refinement of the experimental model. This model can be used for preclinical analysis of both systemic and topical pre-exposure prophylaxis compounds as well as for testing of novel treatments and HIV cure strategies.

We have developed a protocol for the generation and evaluation of a humanized and human immunodeficiency virus-infected NOG mouse model based on stem cell transplant, intravaginal human immunodeficiency virus exposure, and droplet digital PCR RNA quantification.

Ratones NOG humanizados para la exposición y tratamiento del VIH intravaginal

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes ...

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation of human systems, thereby allowing for a more accurate study of pathogenesis, biomarkers, and many other scientific queries. In this method described, immune-deficient NOD-scid IL2R&#947;null (NSG) mice are implanted with autologous thymus, injected with liver-derived CD34+ cells followed by a series of injected cytokine deliveries. In contrast to other models of a similar nature, the model described here promotes an improved reconstitution of immune cells by delivering cytokines and growth factors via transgenes encoded in AAV8 or pMV101 DNA-based vectors. Moreover, it offers long-term stability with reconstituted mice having an average lifespan of 30 weeks after CD34+ injections. Through this model, we hope to provide a stable and impactful method of studying immunotherapy and human disease in a murine model, thus demonstrating the need for predictive preclinical models.

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Humanized mouse models provide a more accurate representation of the human immune microenvironment. This manuscript describes the process in which these models are created through a renal graft of human thymus, injection of human CD34+ cells, and the targeted delivery of human cytokine transgenes to promote CD34+ cell proliferation and differentiation.

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation ...

Oral Combinational Antiretroviral Treatment in HIV-1 Infected Humanized Mice

The human immunodeficiency virus (HIV-1) pandemic continues to spread unabated worldwide, and currently, there is no vaccine available against HIV. Although combinational antiretroviral therapy (cART) has been successful in suppressing viral replication, it cannot completely eradicate the reservoir from HIV-infected individuals. A safe and effective cure strategy for HIV infection will require multipronged methods, and therefore the advancements of animal models for HIV-1 infection are pivotal for the development of HIV cure research. Humanized mice recapitulate key features of HIV-1 infection. The humanized mouse model can be infected by HIV-1 and viral replication can be controlled with cART regimens. Moreover, cART interruption results in a prompt viral rebound in humanized mice. However, administration of cART to the animal can be ineffective, difficult, or toxic, and many clinically relevant cART regimens are unable to be optimally utilized. Along with being potentially unsafe for researchers, administration of cART by a commonly used intensive daily injection procedure induces stress by physical restraint of the animal. The novel oral cART method to treat HIV-1 infected humanized mice described in this article resulted in suppression of viremia below the detection level, increased rate of CD4+ restoration, and improved overall health in HIV-1 infected humanized mice.

This protocol describes a novel method to deliver oral combinational antiretroviral drugs that successfully suppress HIV-1 RNA replication in humanized mice.

Tratamiento antirretroviral combinado oral en ratones humanizados infectados con VIH-1

The human immunodeficiency virus (HIV-1) pandemic continues to spread unabated worldwide, and currently, there is no vaccine available against HIV. ...

Isolating Cord Blood Hematopoietic Stem Cells to Generate Human Immune System Mice

Source: Lanis, J. M., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/64606">Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors.</a> J. Vis. Exp. (2022)
This video demonstrates a technique for generating human immune system (HIS) mice. CD34+ hematopoietic stem cells (HSCs) are isolated from human cord blood buffy coat cells using antibody-coated magnetic beads. The CD34+ HSCs are expanded in a growth medium enriched with cytokines and cryopreserved. Finally, the thawed HSCs are injected into an immunodeficient mouse pup to establish the HIS mouse model.

Aislamiento de células madre hematopoyéticas de la sangre del cordón umbilical para generar ratones del sistema inmunitario humano

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines. All procedures involving animal ...

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Immunology

Immunotherapy

Model Development and Disease Study

Developing a Humanized Mouse Model with Human Liver Cells and Immune Cells

Source: Dagur, R. S. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/58645">Establishment of the Dual Humanized TK-NOG Mouse Model for HIV-associated Liver Pathogenesis.</a>&#160;J. Vis. Exp.&#160;(2019)
This video demonstrates a method for generating a dual humanized mouse model with a human immune system and liver cells. It involves transplanting human liver cells and hematopoietic stem and progenitor cells into a transgenic immunodeficient mouse via intrasplenic injection, leading to engraftment of liver cells and development of human immune cells.

Desarrollo de un modelo de ratón humanizado con células hepáticas humanas y células inmunitarias

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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-Base de células madre Inmunidad Diseñado frente a la infección por VIH en el modelo de ratón humanizado

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