Establishing a murine model for preclinical evaluation of novel erythrocyte transfusion therapies Open Access
Pan, Rachel (Spring 2019)
Published
Abstract
The United States’ blood supply, though large, fails to meet the transfusion needs for certain patient populations, such as those with sickle cell disease and rare blood types. Induced pluripotent stem cell differentiation and expansion may provide a facile way to produce large quantities of patient-personalized, immunologically-compatible red blood cells (RBCs) to augment or replace conventional donated transfusion products. However, before clinical evaluation can take place, stem-derived RBCs must first demonstrate preclinical efficacy and an ability to deliver oxygen to tissues in a manner comparable to conventional transfusion products. This study presents and validates a novel, severely immunocompromised murine model to provide the basis for preclinical studies of novel red blood cell therapies. This model was highly human RBC-tolerant, acutely anemic, and conducive toward direct tissue pO2 measurement. Mice bred on a NOD-SCIDγ background were treated with reagents meant to deplete the innate immune system and given human RBC transfusion. It was demonstrated that the mouse model was highly permissive to human RBCs as transfused cells were found to circulate in vivo up to 42 days. To determine RBCs’ ability to reverse tissue hypoxia and related sequelae, Black 6 mice were made acutely anemic via isovolumic hemodilution using either serum infusion or mouse blood transfusion as fluid replacement. During cycles of bleeding and fluid replacement, changes in tissue pO2 were detected in real time using tissue-localized phosphorescence quenching oximetry. Results indicate that this preclinical model is optimized to demonstrate stem derived-RBCs’ ability to deliver oxygen to tissues and indicate future clinical development, representing a significant step toward clinical applications.
Table of Contents
Introduction 1
Current state and future directions of transfusion therapy in the United States 1
Engineering novel iPSC-derived RBCs for specialized transfusion applications 2
Regulatory obstacles 3
Proposed murine transfusion model 3
Tissue oxygen monitoring 4
Hypothesis, specific aims, strategy, and significance 6
Materials and Methods 7
iPS cell line establishment 7
In vitro hematopoietic differentiation 8
Transfusion-permissive, acutely immunocompromised murine model 9
Flow cytometry 10
Phosphorescence quenching oximetry 12
Results 13
PBMC reprogramming and iPSC differentiation and expansion produced robust yields of terminally mature RBCs 13
NSG mice treated with clodronate liposome, cobra venom factor, and cyclophosphamide were highly tolerant to hRBC transfusion 14
Isovolumic hemodilution effectively induces tissue hypoxia in a facile, quantifiable manner 16
Discussion 18
References 21
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