Scalable Generation of Universal Platelets from Human Induced Pluripotent Stem Cells

Abstract

SummaryHuman induced pluripotent stem cells (iPSCs) provide a potentially replenishable source for the production of transfusable platelets. Here, we describe a method to generate megakaryocytes (MKs) and functional platelets from iPSCs in a scalable manner under serum/feeder-free conditions. The method also permits the cryopreservation of MK progenitors, enabling a rapid “surge” capacity when large numbers of platelets are needed. Ultrastructural/morphological analyses show no major differences between iPSC platelets and human blood platelets. iPSC platelets form aggregates, lamellipodia, and filopodia after activation and circulate in macrophage-depleted animals and incorporate into developing mouse thrombi in a manner identical to human platelets. By knocking out the β2-microglobulin gene, we have generated platelets that are negative for the major histocompatibility antigens. The scalable generation of HLA-ABC-negative platelets from a renewable cell source represents an important step toward generating universal platelets for transfusion as well as a potential strategy for the management of platelet refractoriness.

Generation of HLA-Universal iPSC-Derived Megakaryocytes and Platelets for Survival Under Refractoriness Conditions

“…Based on our studies, we believe that RNAi might be a superior tool to reduce the immunogenicity of iPSC-derived cell products because silencing HLA to upregulate CD62P and to aggregate. These results are in concordance with previous studies, which have shown that iPSC-derived HLA-expressing PLTs are functional (9,39 (B) The graph shows mean and SD of the PLT frequencies detected after HLA-universal MK transfusion in three independent experiments. **p ≤ 0.01.…”

“…Despite some attempts for the establishment of iPSC haplobanks to cover the most frequent HLA haplotypes, it is known from the field of transplantation that even HLA-matched grafts can be rejected due to the presentation of polymorphic mHAs (13,14). Recently, the concept of generating HLA-universal PLTs has been developed using different tools such as RNA interference (RNAi) or Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) (9,10). Previously, we have demonstrated that silencing HLA expression using RNAi prevents an allogeneic immune response in vitro and in vivo.…”

“…It will be difficult to fulfil this necessity only with human volunteer donors (32), therefore, it is desirable to establish alternative PLT sources. To solve the problem of PLT availability, we and others have established different protocols to generate functional PLTs in vitro (9,19,(33)(34)(35). Nevertheless, PLT transfusion is associated with risks and adverse effects.…”

“…In adults, megakaryocytes (MKs) are localized in the bone marrow and extend long protrusions known as proPLTs, through the sinusoidal blood vessels into the circulation. The feasibility to reproduce thrombopoiesis in vitro from pluripotent stem cells has been demonstrated (9,10).…”

Innovation in the field of thrombocytopenias: achievements since the beginning of the century and promises for the future

“…An attractive feature of platelets derived from stem cell culture is the prospect of genetically manipulating progenitor cells to generate platelets devoid of HLA antigens. 12 This would eliminate the risk of alloimmunization against HLA class I antigens and the resulting refractoriness to subsequent platelet transfusions, which is still a serious clinical problem. Clearly, further progress must be made for this platelet source to become a clinical reality.…”