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.

Keywords: GMP-compliant; bioprocess intensification; enucleation; erythropoiesis; erythropoietic transcription factors; genetic amenability; hematopoietic stem cells; human induced pluripotent stem cells; reprogramming; terminal maturation.

Figure 1

Approaches to enhance erythroid differentiation from iPSC. The production of iPSC-derived erythrocytes involves multiple differentiation steps, from mesoderm induction, hematopoietic commitment, erythroid lineage commitment to erythroid differentiation, each identified by specific sets of markers. Different approaches involving genetic modifications or external factors are needed to improve erythropoiesis at each stage. iPSC, induced pluripotent stem cell; HSC, hematopoietic stem cell; Pro-E, proerythroblast; Baso-E, basophilic erythroblast; Poly-E, polychromatic erythroblast; Ortho-E, orthochromatic erythroblast. Created with BioRender.

Figure 2

Scale-up of iPSC-RBC production in a suspension culture. Adapting each stage of the hematopoietic and erythroid specification in the suspension culture provides a starting point to achieve larger volumes and densities. A single 6-well ULA plate (left section) can provide enough cells for expansion and downstream differentiation in increasing volumes, from shake flasks to larger vessels, such as spinner flasks and lab-scale bioreactors (middle section), and eventually transitioning into large, controlled, stirred bioreactors (right section) for high-density erythroid culture and maturation of the cell product. Created with BioRender.