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01.Hematopoietic Stem Cells: Potential New Applications for Translational Medicine
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04.Genetically Engineered Blood Pharming: Generation of HLA-Universal Platelets Derived from CD34+ Progenitor Cells
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Genetically Engineered Blood Pharming: Generation of HLA-Universal Platelets Derived from CD34+ Progenitor Cells $45.00
Authors:  Constança Figueiredo and Rainer Blasczyk
Abstract:
Blood pharming is a recently designed concept to enable in vitro production of blood cells that are safe, effective and readily available. This approach represents an alternative to blood donation and may contribute to overcome the shortage of blood products. However, the high variability of the human leukocyte antigen (HLA) loci remains a major hurdle to the application of off-the-shelf blood products. Refractoriness to platelet (PLT) transfusion caused by alloimmunization against HLA class I antigens constitutes a relevant clinical problem. Thus, it would be desirable to generate PLT units devoid of HLA antigens. To reduce the immunogenicity of cell-based therapeutics, we have permanently reduced HLA class I expression using an RNA interference strategy. Furthermore, we demonstrated that the generation of HLA class I-silenced (HLA-universal) PLTs from CD34+ progenitor cells using an shRNA targeting 2-microglobulin transcripts is feasible. CD34+ progenitor cells derived from G-CSF mobilised donors were transduced with a lentiviral vector encoding for the 2-microglobulin-specific shRNA and differentiated into PLTs using a liquid culture system. The functionality of HLA-silenced PLTs and their ability to escape HLA antibody-mediated cytotoxicity were evaluated in vitro and in vivo. Platelet activation in response to ADP and thrombin were assessed in vitro. The immune-evasion capability of HLA-universal megakaryocytes (MKs) and PLTs was tested in lymphocytotoxicity assays using anti-HLA antibodies. To assess the functionality of HLA-universal PLTs in vivo, HLA-silenced MKs were infused into NOD/SCID/IL-2Rc-/- mice with or without anti-HLA antibodies. PLT generation was evaluated by flow cytometry using anti-CD42a and CD61 antibodies. HLA-universal PLTs demonstrated to be functionally similar to blood-derived PLTs. Lymphocytotoxicity assays showed that HLA-silencing efficiently protects MKs against HLA antibody-mediated complement-dependent cytotoxicity. 80-90% of HLA-expressing MKs, but only 3% of HLA-silenced MKs were lysed. In vivo, both HLA-expressing and HLA-silenced MKs showed human PLT production (up to 0.5% within the PLT population) when anti-HLA antibodies were absent. However, in presence of anti-HLA antibodies HLA-expressing MKs were rapidly cleared from the circulation of mice, while HLA-silenced MKs escaped HLA antibody-mediated cytotoxicity and human PLT production was detectable up to 11 days. Our studies show that HLA-silenced PLTs are functional and efficiently protected against HLA antibody-mediated cytotoxicity. In this chapter, we provide a review of our most recent findings in the use of CD34+ progenitor cells for the production of HLA-universal PLTs and their potential clinical applications. Provision of HLA-universal PLT units may become an important component in the management of patients with PLT transfusion refractoriness. 


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Genetically Engineered Blood Pharming: Generation of HLA-Universal Platelets Derived from CD34+ Progenitor Cells