STEMCELL Technologies
Human hematopoietic cells are generated in waves during development. The first wave produces mainly primitive blood cells that serve the early needs of the growing embryo. Later waves give rise to multipotent hematopoietic stem and progenitor cells (HSPCs), which show adult-like characteristics. While the treatment of hematological malignancies and other blood cell diseases by transplantation of HSPCs from primary tissues, such as bone marrow and peripheral blood, is established, human pluripotent stem cells are an emerging alternative source for generating hematopoietic progenitor cells (HPCs) for similar use. hPSC-derived HPCs offer several advantages over primary HSPCs, including unlimited supply, reduced donor dependency, and compatibility with genetic modification, making them highly promising for the development of therapeutic applications. However, technological challenges such as low yield and limited multilineage potential compared to primary HSPCs need to be addressed before the potential of hPSC-derived hematopoietic therapies can be realized.
In this article, we provide an overview of a highly robust hematopoietic differentiation protocol for generating functional multilineage HPCs in large quantities either in monolayer-based or 3D suspension culture systems. The adaptability of the differentiation protocol allows for its use in screening applications and facilitates manufacture at scale for cell and gene therapy development.
Efficient HPC Generation with Monolayer-Based Differentiation Method
Many hPSC differentiation protocols for generating HPCs and mature blood cells have been described. These protocols, however, lack standardization as they utilize various cytokine combinations and culture lengths, and may require serum, coculture with stromal cells, formation of embryoid bodies (EBs), or ectopic expression of transcription factors (TFs). This results in variable differentiation efficiencies and yields that are highly hPSC-line dependent. The limited multilineage engraftment potential of the cells generated using these protocols also demonstrates that the formation of functional HSPCs is poorly supported. Among the various protocols, 2D monolayer-based methods are generally known to be the most efficient (time- and cost-wise) in generating large numbers of functional CD34+ HPCs with hematopoietic colony-forming and differentiation potential.
One such monolayer-based culture system is the fully optimized and chemically defined STEMdiff™ Hematopoietic Kit, which performs robustly across multiple human embryonic stem (hES) and induced pluripotent stem (iPS) cell lines, with an average yield of 100 CD43+CD34+CD45+ HPCs per seeded hPSC. The kit’s protocol is performed under serum- and feeder-free conditions, does not require expression of potentially oncogenic TFs, and is amenable to the scale-up and development of clinical applications. HPCs generated with the STEMdiff™ Hematopoietic Kit have multilineage differentiation potential, as measured in colony-forming unit (CFU) assays in methylcellulose-based media specifically developed for use with hPSC-derived HPCs.
hPSC-Derived HPCs Have Multilineage Differentiation Potential
hPSC-derived HPCs can also be directed toward specific lineages with lineage-specific growth factors (e.g. EPO, TPO, M-CSF, G-CSF) to generate erythroblasts/erythrocytes, megakaryocytes/platelets, monocytes/macrophages, granulocytes, and microglia.
Erythro-Megakaryocytic Differentiation
Differentiation of hPSCs into erythroid cells enables in vitro red blood cell (RBC) production and offers a potentially unlimited supply of erythroblasts and mature RBCs (also known as erythrocytes) for disease modeling, drug screening, and studying RBC biology. Erythroid differentiation methods may also be scaled up to generate large numbers of RBCs for transfusion. Such hPSC-derived RBCs could become a safe alternative to donor RBCs, e.g. for rare blood types where compatible donor blood is unavailable. Several protocols to generate erythroid cells from PSCs have been developed. These use monolayer-based or EB-formation methods to generate HPCs, followed by downstream differentiation with various EPO-containing cytokine combinations, culture lengths, and media-change schedules.
Using the STEMdiff™ Erythroid Kit, hPSCs can differentiate into erythroid cells, with >70% of cells expressing the erythroid markers CD71 (transferrin receptor) and CD235ab (glycophorin A/B) and a yield of ~10,000 cells per seeded PSC. The hPSC-derived erythroid cells are hemoglobinized and express mainly fetal, some embryonic, and few adult globins based on qPCR and HPLC analyses. Although adult globins are most commonly targeted in gene therapy and the research to enhance their expression remains active, fetal globins are a promising alternative for the treatment of blood disorders, such as sickle cell disease.
Methods have also been developed to promote the differentiation of hPSCs toward megakaryocytes and platelets. Like hPSC-derived RBCs, hPSC-derived megakaryocytes (MKs) and platelets could potentially be used for transfusion, such as to treat patients with thrombocytopenia and alleviate alleviate supply issues due to a shortage of donors and limited shelf-life of platelets from donor blood. The methods described in the field utilize various hPSC seeding methods (single-cell seeding vs. clump seeding), TPO-containing cytokine combinations, and oxygen levels (normoxic vs. hypoxic). Most of these protocols result in low cell yields and generate MKs displaying low ploidy with limited platelet shedding ability. In contrast, the STEMdiff™ Megakaryocyte Kit promotes the differentiation of hPSCs into large, polyploid MKs. The hPSC-derived MKs generated using STEMdiff™ exhibit surface markers CD41a and CD42b (>70% frequency with approximately 300 MKs generated per seeded hPSC), shed platelet-like particles (PLPs; ~3.5 per MK) and, as an indication that they are functional, show increased CD62P surface expression upon stimulation with thrombin and ADP.
Monocyte and Microglia Differentiation
Monocytes are essential components of the innate immune system that provide defense against pathogens or tumors due to their ability to differentiate to macrophages and dendritic cells. While monocytes can be isolated from peripheral blood, hPSCs offer a potentially unlimited source of monocytes that can be used for studying myelopoiesis, disease modeling, drug testing, and developing immune therapies. Both monolayer and EB-formation methods to generate monocytes from hPSCs in differentiation media containing M-CSF have been described. STEMdiff™ Monocyte Kit supports the differentiation of hPSCs to monocytes at an average frequency of 60% CD14+ cells and yield of 100 CD14+ cells per seeded hPSC. The hPSC-derived monocytes can also be further differentiated to dendritic cells and macrophages to produce M1 (classically activated) or M2a (alternatively activated) macrophages in 6- and 8-day culture periods, respectively.
Brain microglia are tissue-resident macrophages that arise predominantly from the first wave of hematopoiesis. hPSC-derived microglia are important tools for modeling neuroinflammation, studying neurological development and disease, co-culture applications, and toxicity testing. Based on the protocol from the Blurton-Jones Laboratory where hPSC-derived CD43+ HPCs are isolated prior to microglial differentiation in media containing M-CSF on matrix-coated plates, STEMCELL Technologies developed a highly efficient protocol to generate and mature microglia coupled with the STEMdiff™ Hematopoietic Kit. The hPSC-derived microglia are highly pure with an average frequency of >80% CD45+CD11b+ cells, >50%TREM2+ cells and <20% of the cells displaying monocyte/macrophage morphology. Similar to hPSC-derived macrophages, STEMdiff™-generated microglia have phagocytic activity (measured by internalization of pH-sensitive bioindicator particles), can be activated in a brain organoid needle injury model, and respond to lipopolysaccharides (LPS) to release proinflammatory cytokines including TNFα, IL-6, IFN-γ, IL-1β, GM-CSF, IL-12p70, IL-2, and IL-8.
Hematopoietic Differentiation Cultures Can be Transitioned Into 3D Suspension Cultures for Scale-Up
In a previous article, we outlined some of the challenges and considerations related to scaling up hPSC cultures, including the differences between scaling up in 2D vs. 3D. As with hPSC cultures, the method of scaling up differentiation protocols depends on multiple factors, including overall goals and desired workflow.
The monolayer-based hematopoietic differentiation protocols described so far typically utilize hPSCs that are maintained in 2D adherent cultures. This 2D culture system can be scaled up using a larger vessel with multiple surface areas, such as cell stacks. However, the cell stack system still requires matrix coating and the cell yield is limited to the surface area of the culture vessels. Besides growing hPSCs as monolayers in 2D adherent culture, high-quality hPSCs can also be grown as aggregates in 3D, matrix-free, suspension culture using appropriate culture vessels (e.g. 6-well plates and Nalgene™ storage bottles) on orbital shakers for 10 to 60 mL culture volumes or in PBS-MINI Bioreactors for larger scales up to 500 mL culture volume (for more information, see our technical manual and protocol). Since hematopoietic cells typically grow in suspension culture, hematopoietic differentiation protocols can be transitioned into 3D suspension culture using the same platforms as for 3D hPSC expansion to streamline the production of HPCs at large scales. HPCs generated in 3D cultures have been found to display similar marker expression, colony-forming potential, and capacity to differentiate into erythroid cells, megakaryocytes/PLPs, and microglia. 3D suspension cultures can provide more efficient differentiation processes when working with larger vessels in high-yield manufacturing workflows. This may be desirable to researchers who require high yields of cells for clinical applications such as the generation of transfusable products or cell therapies or for toxicity testing.
There are key differences between 2D and 3D workflows that may affect growth rates, differentiation kinetics, and how well each hPSC line tolerates shear stress. It may be necessary to optimize critical parameters, such as media change schedules, seeding density, culture time, and mixing speed for successful differentiation in 3D cultures. To assist you, STEMCELL Technologies offers a high-quality PBMC-derived human iPS cell line, SCTi003-A, which could be used as a reference or control line to test and validate differentiation workflows in your lab. SCTi003-A has been validated for use in both 2D and 3D cultures, including in PBS-MINI Bioreactors. This line is also suitable for hematopoietic differentiation using the STEMdiff™ Hematopoietic Kit. If you intend to manufacture HPCs or mature blood cells for clinical cell therapy development, our Services for Cell Therapy team can support your workflows to ensure your products meet your country- or region-specific regulatory requirements.
References
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Li et al. (2023) Modulation of WNT, Activin/Nodal, and MAPK signaling pathways increases arterial hemogenic endothelium and hematopoietic stem/progenitor cell formation during human iPSC differentiation. Stem Cells 41(7): 685-97.
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