Industrially Compatible Transfusable iPSC-Derived RBCs: Progress, Challenges and Prospective Solutions

Abstract

Amidst the global shortfalls in blood supply, storage limitations of donor blood and the availability of potential blood substitutes for transfusion applications, society has pivoted towards in vitro generation of red blood cells (RBCs) as a means to solve these issues. Many conventional research studies over the past few decades have found success in differentiating hematopoietic stem and progenitor cells (HSPCs) from cord blood, adult bone marrow and peripheral blood sources. More recently, techniques that involve immortalization of erythroblast sources have also gained traction in tackling this problem. However, the RBCs generated from human induced pluripotent stem cells (hiPSCs) still remain as the most favorable solution due to many of its added advantages. In this review, we focus on the breakthroughs for high-density cultures of hiPSC-derived RBCs, and highlight the major challenges and prospective solutions throughout the whole process of erythropoiesis for hiPSC-derived RBCs. Furthermore, we elaborate on the recent advances and techniques used to achieve cost-effective, high-density cultures of GMP-compliant RBCs, and on their relevant novel applications after downstream processing and purification.

Generation of red blood cells from stem cells: Achievements, opportunities and perspectives for malaria research

“…However, difficulties associated with variation between lines, persistence of embryonic and/or fetal haemoglobin, low rates of expansion, incomplete differentiation and poor enucleation as well as the complexity of protocols required to differentiate such cells have plagued the quest for iPSC generated red blood cells ( Dias et al., 2011 ; Trakarnsanga et al., 2014 ; Focosi and Pistello, 2016 ). Efforts to improve methodologies for the derivation of such lines and their subsequent differentiation remain a highly active area of research ( Bernecker et al., 2019 ; Hansen et al., 2019 ; Lim et al., 2021 ), however application of such lines for generation of cells suitable for malaria studies so far is extremely limited ( Pance et al., 2021 ).…”

“…Efforts to recapitulate the process of erythropoiesis ex vivo with the ultimate goal of producing in vitro derived red blood cells as a transfusion product have been longstanding and varied. Myriad approaches that address the key obstacles of efficient terminal differentiation and enucleation, sustainability, yield, scalability and cost have been and continue to be developed to this end ( Lim et al., 2021 ; Pellegrin et al., 2021 ). For the malaria research community, enabled access ex vivo to invasion susceptible erythroid cells of increasing interest such as reticulocytes and erythroblasts in quantities amenable to study offers new opportunities for insight.…”

Erythrocyte tropism of malarial parasites: The reticulocyte appeal

“…Answering these questions, however, is extremely challenging considering the diverse cellular differences between reticulocytes and normocytes. Dissecting how individual aspects of the reticulocyte contribute to parasite survival would require a constant homogenous source of reticulocytes (possibly circumvented by using in vitro -generated reticulocytes; Trakarnsanga et al, 2017 ; Lim et al, 2021 ; Sivalingam et al, 2021 ; Yu et al, 2022 ) and methods to experimentally manipulate relevant reticulocyte cellular properties. Regardless, addressing these critical questions is the way forward.…”

Membrane Properties of Human Induced Pluripotent Stem Cell-Derived Cultured Red Blood Cells

“…A promising option for reducing shortages in allogeneic RBC supply is the ex vivo manufacturing of cultured RBC (cRBCs) from human stem cells. Due to their self-renewing properties and their ability to differentiate into all tissues of the human body, induced pluripotent stem cells (iPSCs) offer a very attractive stem cell source for mandatory large-scale production of cRBCs [ 2 , 3 ]. It was estimated that iPSC lines of a few donors would be adequate to cover the requirements of most alloimmunized patients and those with rare blood group phenotypes [ 4 ].…”

“…However, the safety and functionality of cRBC_iPSCs must be thoroughly shown before these cells can be used in clinical or laboratory settings. Thus far, the low efficiency of established culture systems, in terms of expansion and enucleation, have prevented sufficient generation of enucleated cRBC_iPSCs for functional analyses [ 2 , 3 , 5 ]. Especially, the absence of a physiological niche might impair cell growth and lineage-specific differentiation.…”