Human pluripotent stem cells (hPSCs) are an emerging alternative source for generating hematopoietic progenitor cells (HPCs) for applications in disease modeling, drug discovery, and the development of cell and gene therapies for hematological disorders. hPSC-derived hematopoietic cells offer several advantages over primary cells, including an unlimited supply, reduced donor dependency, and compatibility with genetic modification. These features are highly beneficial for drug testing and cell/gene therapy workflows, as evidenced by the use of hPSCs in numerous clinical trials in blood transfusion and chimeric antigen receptor (CAR) immunotherapy.

This technical bulletin provides comprehensive instructions for generating HPCs using a scalable 3D suspension culture system. It covers recommendations on seeding densities, culture conditions, and vessel selection, along with methods for evaluating HPC quality. Additionally, this bulletin discusses data demonstrating the comparability of HPCs generated in the 3D system and the standard 2D monolayer protocol, including efficiency of downstream differentiation into myeloid and erythroid lineage.

Background

During human embryonic development, hematopoietic cells arise through three consecutive and overlapping waves.^1,2 The first wave generates predominantly primitive blood cells, including nucleated erythrocytes and low-ploidy megakaryocytes, which support the early needs of the growing embryo. The second wave produces HPCs, including erythro-myeloid progenitors (EMPs) and lympho-myeloid progenitors (LMPs), which can differentiate into functional blood cells such as erythrocytes, megakaryocytes, and mature myeloid and lymphoid cells. The third wave gives rise to definitive hematopoietic stem cells (HSCs) with multilineage differentiation and long-term engraftment potential, capable of sustaining hematopoiesis throughout adult life. Due to current limitations in understanding developmental precursors and intermediates, as well as the scarcity of HSCs during embryogenesis, most existing hematopoietic differentiation protocols cannot efficiently generate large numbers of engraftable HSCs in vitro.^3-5 Nevertheless, current culture methods can produce HPCs that differentiate into functional, mature blood cells, offering therapeutic potential in areas such as transfusion support, disease modeling, drug screening, and immunotherapy.⁶

Current hPSC hematopoietic differentiation protocols can be broadly categorized into 2D monolayer and 3D embryoid body (EB)-forming suspension approaches. Each method has distinct advantages and limitations.⁷ The 2D protocol is easier to implement, allows quicker adoption, and generates high numbers of floating cells that are easy to harvest. It does not require cell dissociation or specialized equipment such as low-attachment plates. In contrast, the 3D protocol is more amenable to scale-up because cell production is not limited by the surface area of the culture vessel and does not require surface-coating materials. For instance, large-scale vertical-wheel bioreactors occupy a smaller footprint than multilayer cell factories. Recently, an improved 3D approach—the swirling EB method—was introduced. This method allows EBs to continuously release floating CD34+ cells into the culture medium, which are then ready for immediate downstream applications, removing the need for EB dissociation. These CD34+ cells obtained via the swirling EB method have demonstrated long-term engraftment with multilineage reconstitution, marking a major milestone in the field.⁸

STEMdiff™ Hematopoietic Kit (Catalog #05310) efficiently generates HPCs from hPSCs maintained either in 2D adherent culture (in mTeSR™ Plus; Catalog #100-0276/100-1130) or in 3D suspension culture (in TeSR™-AOF 3D; Catalog #100-0720). This differentiation process occurs via an endothelial-to-hematopoietic (EHT) transition in both 2D monolayer and 3D swirling EB suspension cultures (Figure 1). The kit’s chemically defined, animal component-free (ACF) formulation ensures lot-to-lot consistency and reproducible results across diverse hPSC lines, making it suitable for preclinical cell and gene therapy applications.

The following protocol outlines the commonalities and differences between the 2D monolayer and the 3D suspension differentiation methods. It highlights key process parameters for achieving robust hematopoietic differentiation in the 3D system using various culture vessels, including PBS-MINI bioreactors. A comprehensive comparison follows the protocol, evaluating the yield of generated HPCs and their multilineage differentiation efficiency between the standard 2D method (performed in Matrigel®-coated 12-well plates) and the 3D protocol (performed in 6-well plates/Nalgene™ bottles on orbital shakers and in PBS-MINI bioreactors). The downstream differentiation and functionality of the mature progeny, such as microglia, erythroid cells, and megakaryocytes, were also evaluated using the corresponding downstream STEMdiff™ kits. Additionally, the colony-forming potential of the HPCs was evaluated using serum-free MethoCult™ medium.