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)

<ol><li>Karpel, M. E., Boutwell, C. L., Allen, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BLT+humanized+mice+as+a+small+animal+model+of+HIV+infection%5BTitle%5D)+AND+%22Current+opinion+in+virology%22%5BJournal%5D)">BLT humanized mice as a small animal model of HIV infection.</a> Current opinion in virology. 13, 75-80 (2015).</li>
<li>Zhen, A., Kitchen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stem-cell-based+gene+therapy+for+HIV+infection%5BTitle%5D)+AND+%22Viruses%22%5BJournal%5D)">Stem-cell-based gene therapy for HIV infection.</a> Viruses. 6 (1), 1-12 (2014).</li>
<li>Goulder, P. J. R., Watkins, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HIV+and+SIV+CTL+escape%3A+implications+for+vaccine+design%5BTitle%5D)+AND+%22Nature+Reviews%3A+Immunology%22%5BJournal%5D)">HIV and SIV CTL escape: implications for vaccine design.</a> Nature Reviews: Immunology. 4 (8), 630-640 (2004).</li>
<li>Kitchen, S. G., Bennett, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+Antigen-Specific+T+Cells+from+Genetically+Modified+Human+Hematopoietic+Stem+Cells+in+Immunodeficient+Mice%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">Engineering Antigen-Specific T Cells from Genetically Modified Human Hematopoietic Stem Cells in Immunodeficient Mice.</a> PloS one. 4 (12), e8208(2009).</li>
<li>Goulder, P. J. R., Watkins, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HIV+and+SIV+CTL+escape%3A+implications+for+vaccine+design%5BTitle%5D)+AND+%22Nature+Reviews%3A+Immunology%22%5BJournal%5D)">HIV and SIV CTL escape: implications for vaccine design.</a> Nature Reviews: Immunology. 4 (8), 630-640 (2004).</li>
<li>Kitchen, S. G. S., Levin, B. R. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+suppression+of+HIV+by+antigen+specific+T+cells+derived+from+engineered+hematopoietic+stem+cells%5BTitle%5D)+AND+%22PLoS+Pathogens%22%5BJournal%5D)">In vivo suppression of HIV by antigen specific T cells derived from engineered hematopoietic stem cells.</a> PLoS Pathogens. 8 (4), e1002649(2012).</li>
<li>Yang, O. O., Tran, A. C., Kalams, S. A., Johnson, R. P., Roberts, M. R., Walker, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lysis+of+HIV-1-infected+cells+and+inhibition+of+viral+replication+by+universal+receptor+T+cells%5BTitle%5D)+AND+%22PNAS%22%5BJournal%5D)">Lysis of HIV-1-infected cells and inhibition of viral replication by universal receptor T cells.</a> PNAS. 94 (21), 11478-11483 (1997).</li>
<li>Zhen, A., Kamata, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HIV-specific+Immunity+Derived+From+Chimeric+Antigen+Receptor-engineered+Stem+Cells%5BTitle%5D)+AND+%22Molecular+Therapy%22%5BJournal%5D)">HIV-specific Immunity Derived From Chimeric Antigen Receptor-engineered Stem Cells.</a> Molecular Therapy. 23 (8), 1358-1367 (2015).</li>
<li>Barrett, D. M., Singh, N., Porter, D. L., Grupp, S. A., June, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chimeric+Antigen+Receptor+Therapy+for+Cancer%5BTitle%5D)+AND+%22Annual+Review+of+Medicine%22%5BJournal%5D)">Chimeric Antigen Receptor Therapy for Cancer.</a> Annual Review of Medicine. 65 (1), 333-347 (2014).</li>
<li>Pejchal, R., Doores, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Potent+and+Broad+Neutralizing+Antibody+Recognizes+and+Penetrates+the+HIV+Glycan+Shield%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">A Potent and Broad Neutralizing Antibody Recognizes and Penetrates the HIV Glycan Shield.</a> Science. 334 (6059), 1097-1103 (2011).</li>
<li>Caskey, M., Klein, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Viraemia+suppressed+in+HIV-1-infected+humans+by+broadly+neutralizing+antibody+3BNC117%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Viraemia suppressed in HIV-1-infected humans by broadly neutralizing antibody 3BNC117.</a> Nature. 522 (7557), 487-491 (2015).</li>
<li>West, A. P., Scharf, L., Scheid, J. F., Klein, F., Bjorkman, P. J., Nussenzweig, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+insights+on+the+role+of+antibodies+in+HIV-1+vaccine+and+therapy%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Structural insights on the role of antibodies in HIV-1 vaccine and therapy.</a> Cell. 156 (4), 633-648 (2014).</li>
<li>Lan, P., Tonomura, N., Shimizu, A., Wang, S., Yang, Y. -G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reconstitution+of+a+functional+human+immune+system+in+immunodeficient+mice+through+combined+human+fetal+thymus%2Fliver+and+CD34%2B+cell+transplantation%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Reconstitution of a functional human immune system in immunodeficient mice through combined human fetal thymus/liver and CD34+ cell transplantation.</a> Blood. 108 (2), 487-492 (2006).</li>
<li>Melkus, M. W., Estes, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Humanized+mice+mount+specific+adaptive+and+innate+immune+responses+to+EBV+and+TSST-1%5BTitle%5D)+AND+%22Nature+medicine%22%5BJournal%5D)">Humanized mice mount specific adaptive and innate immune responses to EBV and TSST-1.</a> Nature medicine. 12 (11), 1316-1322 (2006).</li>
<li>Shultz, L. D., Brehm, M. A., Garcia-Martinez, J. V., Greiner, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Humanized+mice+for+immune+system+investigation%3A+progress%2C+promise+and+challenges%5BTitle%5D)+AND+%22Nature+Reviews%3A+Immunology%22%5BJournal%5D)">Humanized mice for immune system investigation: progress, promise and challenges.</a> Nature Reviews: Immunology. 12 (11), 786-798 (2012).</li>
<li>Vatakis, D. N., Bristol, G. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+the+BLT+humanized+mouse+as+a+stem+cell+based+gene+therapy+tumor+model%5BTitle%5D)+AND+%22Journal+of+visualized+experiments+%3A+JoVE%22%5BJournal%5D)">Using the BLT humanized mouse as a stem cell based gene therapy tumor model.</a> Journal of visualized experiments : JoVE. (70), e4181(2012).</li>
<li>De Rosa, S. C., Brenchley, J. M., Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Beyond+six+colors%3A+a+new+era+in+flow+cytometry%5BTitle%5D)+AND+%22Nature+medicine%22%5BJournal%5D)">Beyond six colors: a new era in flow cytometry.</a> Nature medicine. 9 (1), 112-117 (2003).</li>
<li>Shimizu, S., Hong, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+highly+efficient+short+hairpin+RNA+potently+down-regulates+CCR5+expression+in+systemic+lymphoid+organs+in+the+hu-BLT+mouse+model%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">A highly efficient short hairpin RNA potently down-regulates CCR5 expression in systemic lymphoid organs in the hu-BLT mouse model.</a> Blood. 115 (8), 1534-1544 (2010).</li>
<li>Denton, P. W., Olesen, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+HIV+latency+in+humanized+BLT+mice%5BTitle%5D)+AND+%22Journal+of+virology%22%5BJournal%5D)">Generation of HIV latency in humanized BLT mice.</a> Journal of virology. 86 (1), 630-634 (2012).</li>
<li>Zhou, J., Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Embryoid+bodies+formation+and+differentiation+from+mouse+embryonic+stem+cells+in+collagen%2FMatrigel+scaffolds%5BTitle%5D)+AND+%22Journal+of+Genetics+and+Genomics%22%5BJournal%5D)">Embryoid bodies formation and differentiation from mouse embryonic stem cells in collagen/Matrigel scaffolds.</a> Journal of Genetics and Genomics. 37 (7), 451-460 (2010).</li>
<li>Vatakis, D. N., Arumugam, B., Kim, S. G., Bristol, G., Yang, O., Zack, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Introduction+of+Exogenous+T-cell+Receptors+Into+Human+Hematopoietic+Progenitors+Results+in+Exclusion+of+Endogenous+T-cell+Receptor+Expression%5BTitle%5D)+AND+%22Molecular+Therapy%22%5BJournal%5D)">Introduction of Exogenous T-cell Receptors Into Human Hematopoietic Progenitors Results in Exclusion of Endogenous T-cell Receptor Expression.</a> Molecular Therapy. 21 (5), 1055-1063 (2013).</li>
<li>Ito, R., Takahashi, T., Katano, I., Ito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Current+advances+in+humanized+mouse+models%5BTitle%5D)+AND+%22Cellular+%26+Molecular+Immunology%22%5BJournal%5D)">Current advances in humanized mouse models.</a> Cellular & Molecular Immunology. 9 (3), 208-214 (2012).</li>
<li>Martinez-Torres, F., Nochi, T., Wahl, A., Garcia, J. V., Denton, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypogammaglobulinemia+in+BLT+humanized+mice--an+animal+model+of+primary+antibody+deficiency%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">Hypogammaglobulinemia in BLT humanized mice--an animal model of primary antibody deficiency.</a> PloS one. 9 (10), e108663(2014).</li>
<li>McCune, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+and+applications+of+the+SCID-hu+mouse+model%5BTitle%5D)+AND+%22Seminars+in+Immunology%22%5BJournal%5D)">Development and applications of the SCID-hu mouse model.</a> Seminars in Immunology. 8 (4), 187-196 (1996).</li>
<li>Srivastava, S., Riddell, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+CAR-T+Cells%3A+Design+Concepts%5BTitle%5D)+AND+%22Trends+in+immunology%22%5BJournal%5D)">Engineering CAR-T Cells: Design Concepts.</a> Trends in immunology. 36 (8), (2015).</li>
</ol>

The authors have nothing to disclose.

Con l&#39;automobile e HSC-based immunità ingegnerizzato acquistando slancio verso gli studi clinici, è importante avere un modello animale adeguato per esaminare da vicino la differenziazione e la funzione di queste cellule ingegnerizzate. In questo protocollo abbiamo descritto i metodi per costruire e testare i topi umanizzati con cellule staminali geneticamente modificate ingegnerizzate contro l&#39;HIV. E &#39;importante avere efficiente trasduzione di cellule staminali prima del trapianto. Tuttavia, a causa della...

Nonostante il successo del combinato terapia anti-retrovirale, l&#39;infezione da HIV è ancora una malattia per tutta la vita. La risposta immunitaria cellulare contro l&#39;HIV gioca un ruolo molto importante nel controllo della replicazione di HIV. I recenti progressi nella manipolazione di cellule staminali ha permesso per il rapido sviluppo di approcci di terapia genica per l&#39;HIV trattamento 1-3. Di conseguenza, è importante avere un modello animale adeguato che permette studio in vivo dell&#39;efficacia di terapie cellulari contro l&#39;HIV. Utilizzo di HIV in modelli animali è complicata dal fatto che il virus infetta soltanto cellule umane. Per aggirare questa limitazione, gli scienziati hanno fatto ricorso all&#39;utilizzo di modelli di malattia come il virus dell&#39;immunodeficienza delle scimmie (SIV) in Rhesus macachi 4,5. Purtroppo, ci sono importanti limitazioni in questo modello a causa delle differenze intrinseche tra le specie e le differenze tra SIV e HIV. Inoltre, solo i servizi altamente specializzati sono capable di sostenere il lavoro con i primati non umani e ogni macaco richiede un grande investimento. Pertanto, vi è una necessità urgente di un modello che utilizza il sistema immunitario umano, che è suscettibile di infezione HIV / patogenesi, ed è meno proibitivo finanziariamente. Il diabetici non obesi (NOD) -Grave combinato immunodeficienti (SCID) -Comune gamma catena knockout (c γ - / -) (o NSG) Sangue / Fegato / Thymus (BLT) modello di topo umanizzato è sempre dimostrato di essere uno strumento importante per studiare l&#39;infezione da HIV. Impiantando cellule staminali ematopoietiche (CSE) e del timo fetale, i topi sono in grado di sviluppare e riepilogare un sistema immunitario umano 1-3. Un tipo di terapia genica basata sulle cellule staminali coinvolge &#39;reindirizzamento&#39; cellule T periferiche di indirizzare l&#39;HIV riprogrammando cellule staminali ematopoietiche (CSE) di differenziarsi in cellule T antigene specifico. Abbiamo dimostrato in precedenza che HSC ingegneria con un molecolare clonato anti-HIV specifica re delle cellule Trecettore (TCR) contro l&#39;epitopo SL9 (aminoacido 77-85; SLYNTVATL) del virus HIV-1 gag può reindirizzare le cellule staminali in formazione di linfociti T maturi che sopprimono la replicazione di HIV nel umanizzato NSG-BLT modello di topo 6. L&#39;avvertimento di utilizzare un TCR clonato molecolare è che è limitato ad uno specifico sottotipo antigene leucocitario umano (HLA) che limitare l&#39;applicazione di questa terapia. recettori chimerici antigene (CAR), d&#39;altro canto, possono essere universalmente applicati a tutti i sottotipi HLA. Gli studi iniziali sono stati eseguiti utilizzando un CAR costruito con i domini extracellulari e transmembrana di CD4 umana fusa alla ζ segnalazione intracellulare dominio di CD3 (definito il CD4ζCAR). CD4ζCAR espresso sulle cellule T CD8 può riconoscere busta HIV e scatenare una risposta delle cellule T citotossiche che è simile a quella mediata da un recettore delle cellule T 7. Abbiamo recentemente dimostrato che cellule staminali emopoietiche umane possono essere modificati con CD4ζCAR, che possono poi differenziarsi in molteplici ematopoietiche Lineages, comprese le cellule T funzionali in grado di sopprimere la replicazione dell&#39;HIV nel modello umanizzato del mouse 8. Con il rapido avanzamento in chimerici terapie recettore antigene per il cancro 9, e la caratterizzazione continuo di potenti ampie anticorpi neutralizzanti 10-12 contro l&#39;HIV che permettono la costruzione di un&#39;unica catena CAR anticorpi, è percepibile che molti nuovi costrutti candidati, in aggiunta a CD4ζCAR , sarà generato e testato per la terapia genica a base di cellule staminali di malattie HIV e altre malattie. Inoltre, il modello di topo NSG-BLT umanizzato contenente queste vetture antigene-specifiche può anche fornire un utile strumento per esaminare attentamente le risposte delle cellule T umane in vivo. È importante sottolineare che il nostro protocollo differisce dai metodi descritti precedenti per la costruzione di umanizzato di topi BLT 13-15 dal fatto che la CSE in miscela proteica gelatinosa viene utilizzato al posto di tronchi fegato fetale 16. Questo protocollo descrive: 1) costruzione di Humanitopi BLT zed ingegnerizzati con CD4ζCAR; e 2) caratterizzazione del differenziamento delle cellule geneticamente modificate; e 3) caratterizzazione della funzionalità delle cellule geneticamente modificate.

<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 il rapido sviluppo di terapie geniche a base di cellule staminali contro l&#39;HIV, non vi è necessità di premere un modello animale per studiare la differenziazione ematopoietiche e la funzione immunitaria delle cellule geneticamente modificate. Il umanizzato midollo osseo / Fegato / Thymus (BLT) topo modello consente la piena ricostituzione di un sistema immunitario umano in periferia, che comprende le cellule T, cellule B, cellule NK e monociti. L&#39;impianto del timo umano consente anche per la selezione timica delle cellule T nel tessuto del timo autologo. Oltre allo studio dell&#39;infezione da HIV, il modello rappresenta un potente strumento per studiare la differenziazione, lo sviluppo e la funzionalità delle cellule derivate da cellule staminali ematopoietiche (CSE). Qui descriviamo la costruzione di umanizzato diabetici non obesi (NOD) -Grave combinato immunodeficienti (SCID) -Comune gamma catena knockout (c γ - / -) topo Hong osseo osseo / Fegato / Thymus (NSG-BLT) con CSE trasdotte con CD4 recettore per l&#39;antigene chimerico (CD4CAR)vettore lentivirus. Abbiamo dimostrato che il CD4CAR CSE può differenziarsi con successo in molteplici linee e hanno attività anti-HIV. L&#39;obiettivo dello studio è quello di dimostrare l&#39;uso del modello di topo NSG-BLT come un modello in vivo per l&#39;immunità ingegnerizzato contro l&#39;HIV. Vale la pena notare che, poiché viene utilizzato lentivirus e tessuti umani, esperimenti e interventi chirurgici devono essere eseguiti in una classe II armadio biosicurezza in un livello di biosicurezza 2 (BSL2) con particolari precauzioni (BSL2 +) struttura.

La figura 1 mostra uno schema di costruzione di topi BLT umanizzati con cellule staminali modificato. 10 settimane dopo la chirurgia implantare, i topi sono stati sacrificati per valutare la differenziazione e sviluppo di cellule geneticamente modificate. Come mostrato in figura 2, molteplici tessuti linfoidi (sangue, milza, timo e midollo osseo) sono state raccolte da un topo che è stato modificato con CD4ζCAR. Il CD4ζCAR utilizzato in questo protoco...

Watch this Scientific Journal Video about Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model at JoVE.com

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.

Cellule staminali base Immunità Engineered contro l&#39;infezione da HIV nel modello umanizzato mouse

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

stem-cell-based-engineered-immunity-against-hiv-infection-humanized

Research

JoVE Journal

Medicine

10.5K Views.  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 mouse BLT umanizzato come cellule staminali basata su un Modello Tumore Terapia Genica

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

Ricerca

Immunologia e infezioni

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.

Test di immunoterapie del cancro in un modello murino umanizzato che porta tumori umani

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

Ricerca sul cancro

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.

Umanizzato NOD/SCID/IL2rγnull (hu-NSG) Mouse modello per studi di latenza e di replicazione dell'HIV

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.

Istituzione del modello murino TK-NOG dualizzato per la patogenesi del fegato associata all'HIV

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.

Infezione da HIV cronica, acuta e riattivata in modelli di topo immunodeficienti umanizzati

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.

Mitrai NOG umanizzati per l'esposizione all'HIV intravaginale e il trattamento dell'infezione da HIV

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.

<meta charset="utf8"></head><body>Produzione di topo umanizzato tramite innesto di capsula renale timica, iniezione di cellule CD34+ e somministrazione di citochine

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.

Produzione di topo umanizzato tramite innesto di capsula renale timica, iniezione di cellule CD34+ e somministrazione di citochine

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.

Trattamento antiretrovirale combinato orale in topi umanizzati infetti da HIV-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/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.

Isolamento delle cellule staminali ematopoietiche del sangue del cordone ombelicale per generare topi del sistema immunitario umano

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

Cellule staminali base Immunità Engineered contro l'infezione da HIV nel modello umanizzato mouse

Riepilogo

Esplora Altri Video