Immune cells and RBCs derived from human induced pluripotent stem cells: method, progress, prospective challenges

Abstract

Blood has an important role in the healthcare system, particularly in blood transfusions and immunotherapy. However, the occurrence of outbreaks of infectious diseases worldwide and seasonal fluctuations, blood shortages are becoming a major challenge. Moreover, the narrow specificity of immune cells hinders the widespread application of immune cell therapy. To address this issue, researchers are actively developing strategies for differentiating induced pluripotent stem cells (iPSCs) into blood cells in vitro. The establishment of iPSCs from terminally differentiated cells such as fibroblasts and blood cells is a straightforward process. However, there is need for further refinement of the protocols for differentiating iPSCs into immune cells and red blood cells to ensure their clinical applicability. This review aims to provide a comprehensive overview of the strategies and challenges facing the generation of iPSC-derived immune cells and red blood cells.

Keywords: iPSC (induced pluripotent stem cell); iPSC dereved iNKT cell; iPSC derivation; iPSC derived NK cell; iPSC derived T cell; iPSC derived macrophages cell; red blood cell.

FIGURE 1

Strategies for producing iPSC-derived T cells. Hematopoietic differentiation can be initiated using various protocols, including feeder-free methods like monolayer systems, co-culture with mouse stromal cells, and EB formation. At this stage, endothelial-to-hematopoietic transition, CD34⁺ HSPCs emerge from the HE layers. The specification of T cell lineage requires Notch signaling, which can be made easier by co-culture with mouse stromal cells such as OP9-DL1 or OP9-DL4. Mature T cells can be efficiently generated by co-culturing iPSC-derived multipotent HSPCs with these cells in a 2D or 3D system. A coating matrix mixture that contains retronectin and recombinant DL4 protein can be used to provide Notch signals as an alternative.

FIGURE 2

Strategies to generate iPSC-derived NK cells. iPSCs are induced to initiate hematopoietic differentiation through co-culturing with mouse feeder cells (M210-B4 cells) or by forming embryoid bodies (EB). During this stage, CD34⁺ HSPCs emerge from the HE layers, similar to the protocol for iPSC-derived T cells. The specification of NK cell lineage is then achieved either through a feeder-dependent system or a feeder-independent system.

FIGURE 3

Strategies of iPSC-derived macrophage cells generation in OP9-independent protocol. The differentiation of macrophages derived from iPSC has 4 main stages. (1) mesoderm/HE induction, (2) Hematopoietic differentiation, (3) Myeloid specification, (4) terminal differentiation of macrophages derived from iPSC differentiation. Mesoderm/HE induction are generated by 2D-F protocol or 3D protocol. In 2D-F protocol, iPSCs are cultured in M atrix-coated plates with complex mixes of exogenous factors to driver cells differention. In 3D protocol, the step is completed by EB formation. Based on the presence or absence of exogenous factors, it is divided into EB-S protocol and EB-F protocol. In the 2D-F protocol, a series of exogenous factors with complex compositions is introduced in a sequential manner to orchestrate the progression of cells towards hematopoietic progenitor formation and subsequent myeloid specification. EBs are initially generated and subsequently transferred onto tissue culture plates using the EB-S protocol. These EBs are then cultured with interleukin-3 (IL-3) and macrophage colony-stimulating factor (M-CSF), which synergistically induce the emergence of hematopoietic progenitors and their directed commitment towards the myeloid lineage. Alternatively, the EB-F protocol allows for EB induction either through IL-3 and M-CSF supplementation or through the strategic utilization of a composite mixture comprising various exogenous factors.