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

摘要

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.

摘要

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 IL2Rγnull (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.

引言

While animal models have created a deeper understanding of cellular and molecular systems, the challenge remains in elucidating the intricacies of species-specific systems, such as immunity, physiology, and other areas of pathology. Non-human primates (NHP), such as chimpanzees, have historically been used to compensate for the wanting gaps in model research; however, the NHP model can be quite costly and inaccessible, particularly as their use has been banned in Europe¹.

Following a successful grafting procedure, the murine system replicates the human immune system, as demonstrated through the repopulation of the ly....

研究方案

All protocols involving the use of animals are closely monitored by the Wistar Institute's Institutional Animal Care and User Committee (IACUC). The laboratory adheres to the guidelines set by this committee and the attending veterinarian to ensure the health, safety, and wellbeing of the animals involved. Prior to following this protocol, veterinarian and IACUC approval are required, and individuals may have variations in the specific surgical techniques and animal handling compared to the protocol per the advice of those aforementioned parties involved in animal welfare.

NOTE: Tissue samples can be frozen until ready for use. Addition....

结果

Following successful surgery and appropriate postoperative injections, CD34⁺ differentiation can be confirmed via flow cytometry. Approximately 8 weeks after surgery, mice are bled in preparation for FACS, recurring every 2 weeks until a specific threshold of human immune cells is met as is described previously¹⁰. Briefly, 100 mL of blood was collected in blood collection tubes coated with lithium and heparin. After the lysis of red blood cells using ACK lysis buffer, the cells were was.......

讨论

This manuscript has herein described generating humanized mice via human fetal thymus grafted under the renal capsule and subsequent CD34⁺ injection to recreate a human immune system.

While the protocol functions to create the best model possible, certain steps are essential to viability. For example, during the CD34⁺ isolation, it is essential that one looking through the microscope can identify CD34⁺ cells. Though it may seem redundant, automatic counting mac.......

材料

Name	Company	Catalog Number	Comments
ACK lysis buffer	Life Technologies Corporation
BD Microcontainer	BD		blood collection tubes
Busulfan	Sigma	B2635-25g	Irradiating drug; light sensitive
CD34 Microbeads	Miltenyi Biotec	130-046-702	antibody beads kit; stored at 4 °C
CKIT	Aldevron	custom	cytokine; stored at -20 °C
Collagenase/Dispase	Roche Diagnostics	11097113001	Stored at 4 °C
FcR Blocking reagent	Miltenyi Biotec	130-046-702	antibody beads kit; stored at 4 °C
Fetal tissue (liver and thymus)	Advanced Bioscience Resources		Delivered same day or overnight
Ficoll	GE Healthcare	17-1440-03	Stored at room temperature
FLT3	Aldevron	125964	cytokine; stored at -20 °C
Forceps	Various	Various
Hamilton syringe needle	Various	Various	22 G; 3 point; 2" length
Hemostats	Various	Various
MS columns	Miltenyi Biotec	130-042-201	magnetic separator
PBS	Gibco	14190-136	Stored at room temperature
Primocin	Invivogen	amt-pm1	antibiotic; stored at 4 °C
RPMI	Corning	10-040-CM	Stored at 4 °C
SCF	Aldevron	125962	cytokine; stored at -20 °C
Surgical scissors	Various	Various
THPO	Aldevron	125963	cytokine; stored at -20 °C
Tissue treated petri dish	Corning	430167
VetBond glue	3M	1469SB	glue
Visorb suture	Stoelting Co	5046	absorbable suture, size 4, 19 mm cutting