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.

CAR ve klinik çalışmalarda doğru HSC tabanlı mühendislik bağışıklık kazanıyor ivme ile, yakından bu mühendislik hücrelerin farklılaşması ve işlevini incelemek için uygun bir hayvan modeli olması önemlidir. Bu protokolde inşa ve HIV&#39;e karşı tasarlanmış genetiği değiştirilmiş kök hücrelerle hümanize fareler test edilmesi için yöntemler tarif. Nakli öncesinde kök hücrelerin etkin transdüksiyonunu olması önemlidir. Bununla birlikte, hedef hücrelerin tanınması üzerine çoğalma T hücresi yeteneği nedeniyle, kök hücre modifikasyonunun düşük seviyelerde, HIV replikasyonunun 8&#39;e karşı güçlü bir tepki elde etmek için yeterli idi. Bununla birlikte, kök hücre değişikliğinin yüksek düzeyde sağlamak için, otolog timus yerine karaciğer ve başka bir yerde 13-15 tarif edilmişti fareler ameliyat için timus parçaları ile jelatinimsi protein karışımı içinde transduced CD34 + hücreler kullanmanızı öneririz. Jelatinimsi Protein karışımı solubiliz olanhücre dışı matriks protein açısından zengin ed doku bazal membran. Normal fizyolojik şartlar altında, jelatinimsi bir protein karışımı, kök hücre 20 etkin eki ve farklılaşmasını sağlayacak bir yeniden, biyolojik olarak aktif ve stabil matrisi üretmek üzere polimerize. İnsan hücre sulandırma retroorbital kanama kontrol ve 6 akım sitometri olabilir - 8 hafta sonrası cerrahi. Vektör kök hücre hayatta kalma ve yenilenmesi için toksik olmayan sağlamak için, 1.2.6 tarif edildiği gibi deney öncesinde CD34 + hücrelerinde vektör titre önerilir. NTG-blt fare modeli kapasitesi, HIV enfeksiyonunu tedavi etmek için 1 HIV bağışıklık patolojisi ve hücre bazlı terapiler incelemek için yüksek ölçüde yararlı bir model haline getirir mukoza enfeksiyonu tutarlı viremi ve hücresel bağışıklık tepkilerini desteklemek. En önemlisi, NSG-BLT farelerden elde T hücreleri kaderi incelemek için araştırmacı izin otolog timik dokusunda seçilirtimik seçimi 8,21 sonra gen modifiye kök hücrelerin. açıklanan yöntemle, sürekli 40% -90% insan immün hücre sulandırma almak mümkün olmuştur. insan hücre sulandırma düşük seviyelerde cerrahi ve organ nakli için doku kalitesini yapan kişinin beceri dahil olmak üzere birçok faktöre kaynaklanabilir. insan hücre sulandırma yüksek seviyede elde edilmesi için, güvenli bir şekilde böbrek kapsülü altından yerleştirilen nakli sağlamak için önemlidir. Buna ek olarak, son derece hafif mikroskop altında ameliyat için hazırlanan her timik implantı incelemek ve herhangi bir şüpheli parçaları atmak için tavsiye edilir. Insanlaştırılmış blt fare modelinde (1,15,22 gözden) HIV&#39;e karşı tasarlanmış bağışıklık çalışmak için gelecek vaat eden bir araç da, kendi sınırlamaları vardır. Yani, bu model mükemmel bir insan periferal bağışıklık sistemini taklit etmez. Çalışmalar hiper-mutasyona uğramış, sınıf-anahtarlamalı IgG Antibo bozukluğu gelişme göstermiştir1,23 dy. Ayrıca, bağışıklık eksikliği fareler kullanılarak teknik açıdan zor ve sağlıklı bu fareler tutmak önemli kaynaklar ve eğitim gerektirir. Ince fırsatçı enfeksiyonlar numuneler üzerinde olduğu gibi önemli farklılıklar tezahür ve potansiyel deney üzerinde olumsuz sonuçlar doğurabilir. Nedenle gelecekteki veri 1,24 bütünlüğünü korumak için iyi hazırlanmış tesisleri ve uygun şekilde eğitilmiş personel olması önemlidir. Bu örneklerde 4,8 gösterildiği gibi kafasında böyle sınırlamalar, insanlaştırılmış NSG-BLT fare modeli hala kök hücre bazlı mühendislik bağışıklığın çalışma için önemli bir araç sağlar. Antikorlar 10 ve daha verimli CAR 25 sinyal etki modifikasyonunu nötralize HIV Broad dayalı kimerik antijen reseptörleri geliştirme eğilimi ile, bu model ve protokol karakterize ve gen modifiye cel işlevselliğini araştırmak için kullanılabilirSYR&#39;lerinden yeni nesil ls. Buna ek olarak, bu model potansiyel projelendirilmiş bağışıklık birlikte (örneğin inhibitör reseptör blokajı gibi) bağışıklık bazlı terapi çalışmaları ağırlayacak.

Kombine antiretroviral tedavinin başarısına rağmen, HIV enfeksiyonu hala hayat boyu bir hastalıktır. HIV&#39;e karşı hücresel immün yanıt HIV çoğalmasını kontrol etmede oldukça önemli bir rol oynamaktadır. Kök hücre manipülasyonu son gelişmeler HIV tedavisi 1-3 yaklaşımlar gen tedavisinin hızlı gelişimi için izin vermektedir. Bunun bir sonucu olarak, HIV&#39;e karşı hücre bazlı terapilerin etkinliğinin in vivo çalışmada sağlayan bir uygun hayvan modeli olması önemlidir. hayvan modellerinde HIV ile çalışan virüs sadece insan hücreleri enfekte olduğu gerçeği ile karmaşık. Bu sınırlama aşmak için, bilim adamları Rhesus makak 4,5 yılında Simian İmmün Yetmezlik Virüsü (SIV) gibi hastalık modelleri kullanarak başvurdular. Ne yazık ki, türler arasında doğal farklılıklar ve SIV ve HIV arasındaki farklılıklar, bu modelde temel sınırlamalar vardır. Buna ek olarak, sadece son derece uzmanlaşmış tesisleri c vardırinsan olmayan primatlar ve her makak ile çalışmalarını destekleme apable büyük bir yatırım gerektirir. Böylece, HIV enfeksiyonu / patogenezi duyarlı insan bağışıklık sistemini kullanır ve daha az mali yasaklayıcı bir model için acil bir ihtiyaç vardır. (Ya da NTG) Kan / Karaciğer / Thymus (BLT) insanlaştırılmış fare modeli giderek olduğu kanıtlanmıştır önemli bir araç obez olmayan diyabetik (NOD) kombine immün yetmezliği olan (SCID) -ortak gama zinciri nakavt - (- / c γ) -severe HIV enfeksiyonunu incelemek için. Hematopoetik kök hücreleri (HKH&#39;lerin) ve fetal timus implante ederek, fareler geliştirmek ve insan bağışıklık sistemini 1-3 recapitulate edebiliyoruz. kök hücre bazlı gen terapisinin bir tipi, antijene özel T hücreleri ayırt etmek Hematopoietik kök hücreler (HSC), yeniden programlayarak HIV hedef periferik T hücrelerinin yönlendirme &#39;içerir. Daha önce göstermiştir ki, bir molekül klonlanmış bir anti-HIV spesifik T hücresi yeniden mühendislik HSCSL9 epitopa (amino asit 77-85; SLYNTVATL) karşı reseptör (TCR), HIV-1 Gag hümanize NTG-blt fare modelinde 6 HIV kopyalanmasını bastırdığı Olgun T hücreleri oluşturan kök hücre yeniden yönlendirebilir. molekül klonlanmış TCR kullanılarak ikaz bu tedavinin uygulanmasını sınırlar, belirli bir insan lökosit antijeni (HLA) alt tipi ile sınırlı olmasıdır. Kimerik antijen reseptörleri (CAR), diğer taraftan, evrensel olarak tüm HLA alt tiplerinin uygulanabilir. İlk çalışmalar CD3 alan sinyal hücre içi ζ birleştirilmiş insan CD4, hücre dışı ve transmembran ile inşa bir araba kullanılarak yapıldı (CD4ζCAR olarak da adlandırılır). CD4ζCAR HIV zarf tanır ve bir T hücresi reseptörü tarafından aracılık edilen 7 edilene benzer bir sitotoksik T hücre yanıtını tetikleyen CD8 T hücreleri üzerinde ifade edilmiştir. Biz son zamanlarda insan HKH&#39;lerin sonra birden hematopoetik li içine ayırt edebilirsiniz CD4ζCAR ile modifiye edilebilir olduğunu göstermiştirinsanlaştırılmış fare modelinde 8 HIV replikasyonunu bastırma yeteneğine sahip fonksiyonel T hücreleri de dahil olmak üzere neages. Kanser 9 kimerik antijen reseptör terapileri hızlı ilerlemesi ve güçlü geniş nötralize antikorları tek zincirli antikor SYR&#39;lerinden inşaatına izin HIV&#39;e karşı 10-12 sürekli karakterizasyonu ile, algılanabilir CD4ζCAR yanı sıra pek çok yeni aday yapıları olduğunu oluşturulan ve HIV hastalıklar ve diğer hastalıkların kök hücre bazlı gen terapisi için test edilecektir. Buna ek olarak, bu antijene özgü kabinli insanlaştırılmış NTG-blt fare modeli de yakından in vivo olarak insan T hücresi yanıtlarını incelemek için yararlı bir araç sağlayabilir. Önemli olarak, protokol BLT fareler, HSC jelatinimsi protein karışımı fetal karaciğer gövdeleri 16 yerine kullanılır 13-15 hümanize yapımı için bir önceki açıklanan yöntemlere farklıdır. Bu protokol açıklar: humani 1) inşaatıCD4ζCAR ile mühendislik zed BLT fareler; ve 2) genetiği değiştirilmiş hücrelerin farklılaşma karakterizasyonu; genetik olarak tadil edilmiş hücrelerin işlevselliği ve 3) karakterizasyonu.

<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

HIV&#39;e karşı kök hücreye dayalı gen terapisi hızlı gelişimi ile, genetik olarak tadil edilmiş hücrelerin hematopoietik farklılaşmasını ve bağışıklık fonksiyonu incelemek için bir hayvan modeli için gereksinimi basarak vardır. İnsanlaştırılmış kemik iliği / Karaciğer / Timus (BLT) fare modeli T hücreleri, B hücreleri, NK hücreleri ve monositler dahil çevrede, bir insan bağışıklık sistemi tam sulandırma sağlar. insan timus implant, aynı zamanda, otolog timik dokuda T hücrelerinin timus seçimine izin verir. HIV enfeksiyonu çalışma ek olarak, model, hematopoietik kök hücreler (HSC), elde edilen hücrelerin farklılaşması, geliştirme ve özelliğe araştırmak için güçlü bir araç konumundadır. Kalıt HSCler ile -Bone iliği / Karaciğer / Timus (NTG-BLT) fareler ile Burada kombine bağışıklık eksikliği olan (SCID) -Ortak gama zinciri nakavt - (- / C γ) -severe insanlaştırılmış obez olmayan diyabetik (NOD) yapımına özetlemektedir CD4 kimerik antijen reseptörü ile (CD4CAR)lentivirüs vektör. Bu CD4CAR HSC başarıyla birden fazla soy dönüştürmek ve anti-HIV etkinliğe sahip olduğunu göstermektedir. Çalışmanın amacı, HIV&#39;e karşı tasarlanmış bağışıklık için bir in vivo modeli olarak NTG-blt fare modelinde kullanımını göstermektir. Bu lentivirüs ve insan dokusu kullanıldığı için, deneyler ve ameliyatlar özel önlemler (BSL2 +) tesisi ile Biyogüvenlik Seviyesi 2 (BSL2) bir Sınıf II biyogüvenlik kabini yapılmalıdır, dikkati çekiyor.

Şekil 1 değiştirilmiş kök hücre ile insanlaşmış BLT farelerin inşa bir taslağıdır. 10 hafta implant cerrahisi sonrası, fareler gen modifiye edilmiş hücrelerin farklılaşmasını ve gelişmesini değerlendirmek için feda edildi. Şekil 2&#39;de gösterildiği gibi, birden fazla lenfoid dokular (kan, karaciğer, dalak, timus, kemik iliği) CD4ζCAR ile değiştirilmiş olan bir fareden hasat edilmiştir. Bu protokolde kullanılan CD4ζCAR anti-CD4 antikoru ve GFP 8 sentezlenmesi ile tespit edilebilir CD4 kimerik antijen reseptörü ve GFP içerir. Hücreler izole edilmiş ve, insan CD45 karşı antikor ve anti-CD4 antikoru ile boyanmış ve akış sitometrisi ile analiz edilmiştir. GFP ve CD4 çift pozitif hücrelerin birden çok lenfoid dokularda CD4CAR + hücrelerinin mevcudiyetine işaret eden, tespit edilmiştir. Gen modifiye edilmiş hücrelerin farklılaşmasını araştırmak için,<html> Splenositler, insan CD45 (lenfosit), CD3 (T hücreleri), CD19 (B hücreleri), CD14 (monosit ve makrofajlar) ve CD337 (NK hücreleri) karşı antikorlar ile boyandı. Şekil 3&#39;te gösterildiği gibi, CD4ζCAR hematopoietik kök hücreler birden fazla soy içinde farklılık gösterir. CD4CAR değiştirilmiş hücreler fonksiyonel olup olmadığını araştırmak için, biz CD4CAR hücreleri (HIV bulaşmış T1 hücreleri veya kontrol olarak bulaşmamış T1 hücreleri) tanıyacağını hedef hücreleri ile splenositlerin coincubated. Hücreler, gece boyunca birlikte inkübe edildi ve protein taşınma inhibitörünün ilave 6 saat boyunca ilave edildi. Daha sonra, hücreler, sabit ve IFNy ve TNFa gibi sitokinlerin hücre içi ifadesi için leke permeabilize edilmiştir. Şekil 4&#39;te gösterildiği gibi, ARAÇ ifade eden hücreler, enfekte T1 hücreleri ile IFNy ve TNFa yüksek miktarda üretilir. yük / 54048 / 54048fig1.jpg &quot;/&gt; Şekil 1:. Fetal timus: değiştirilmiş kök hücrelerle insanlaşmış BLT farelerin inşaat FT Anahat. FL:. Fetal karaciğer <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54048/54048fig1large.jpg" target="_blank">bu rakamın daha büyük bir versiyonunu görmek için lütfen buraya tıklayınız.</a> <img alt="şekil 2" src="/files/ftp_upload/54048/54048fig2.jpg" /> Şekil 2:., CD4 kimerik antijen reseptör modifiye edilmiş hücreler çok lenfoid dokularda tespit edilebilir fare CD4CAR değiştirilmiş HSCler ile toplandı cerrahi ve birden çok lenfoid dokuların 10 hafta sonra kurban edildi ve hücreler, anti-insan CD45 ve anti-insan CD4 antikor ile boyandı ve flow sitometri ile analiz edilmiştir. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54048/54048fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> Şekil 3:.. CD4 kimerik antijen reseptörü değiştirilmiş hücreler birden fazla soy içine ayırt edebilirsiniz CD4CAR değiştirilmiş farelerin splenositler hasat ve insan CD45, CD3, CD19, CD14 ve CD337 karşı antikorlar ile boyandı ve akış ile analiz edilmiştir sitometri <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54048/54048fig3large.jpg" target="_blank">bir büyük görmek için tıklayınız Bu rakamın sürümü.</a> <img alt="Şekil 4," src="/files/ftp_upload/54048/54048fig4.jpg" /> Şekil 4:. CD4 CAR + T hücrelerinin ex vivo sitokin tahlil HIV elde edilen dalak CD4CAR fareler gösterilir HIV bulaşmış veya enfekte olmayan T1 hücreleri ve sitokin onların hücre içi üretimi ya ile uyarıldı enfekte. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54048/54048fig4large.jpg" target="_blank">Vie için tıklayınız</a>Bu rakamın wa daha büyük bir versiyonu.

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.

Insanlaşmış Fare Modelinde HIV enfeksiyonuna karşı kök hücre Tabanlı Engineered Bağışıklık

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

Nanyang Technological University

Research

JoVE Journal

Medicine

10.1K Görüntüleme.  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.

Bir Kök Hücre tabanlı Gen Terapisi Tümör Modeli olarak BLT Insanlaşmış Fare Kullanma

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

İmmünoloji ve Enfeksiyon

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.

İnsan Tümörleri Taşıyan İnsanlaştırılmış Bir Fare Modelinde Kanser İmmünoterapötiklerinin Test Edilmesi

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

Kanser Araştırmaları

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.

İnsanlaşmış NOD/SCID/IL2rγnull (hu-NSG) fare modeli HIV çoğaltma ve gecikme süresi çalışmaları için

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.

HIV ile ilişkili Karaciğer Patogenezi için Çift İnsanize TK-NOG Fare Modelinin Oluşturulması

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.

Humanized Immunoficient Mouse Modellerinde Kronik, Akut ve Yeniden Aktive HIV Enfeksiyonu

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.

Intravajinal HIV Maruziyeti ve HIV Enfeksiyonu Tedavisi için İnsanize NOG Fareler

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.

HIV-1 Enfekte İnsanlaştırılmış Farelerde Oral Kombinasyonel Antiretroviral Tedavi

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.remotexs.ntu.edu.sg/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.

İnsan Bağışıklık Sistemi Fareleri Oluşturmak için Kordon Kanı Hematopoetik Kök Hücrelerinin İzole Edilmesi

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

JoVE Encyclopedia of Experiments

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Model Geliştirme ve Hastalık Çalışması

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

Source: Dagur, R. S. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/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.

İnsan Karaciğer Hücreleri ve Bağışıklık Hücreleri ile İnsanlaştırılmış Bir Fare Modeli Geliştirme

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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Insanlaşmış Fare Modelinde HIV enfeksiyonuna karşı kök hücre Tabanlı Engineered Bağışıklık

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