As a standard clinical treatment, platelet transfusion has been employed to prevent hemorrhage in patients with thrombocytopenia or platelet dysfunctions. Platelets also show therapeutic potential for aiding liver regeneration and bone healing and regeneration and for treating dermatological conditions. However, the supply of platelets rarely meets the rising clinical demand. Other issues, including short shelf life, strict storage temperature, and allogeneic immunity caused by frequent platelet transfusions, have become serious challenges that require the development of high-yielding alternative sources of platelets. Human pluripotent stem cells (hPSCs) are an unlimited substitution source for regenerative medicine, and patient-derived iPSCs can provide novel research models to explore the pathogenesis of some diseases. Many studies have focused on establishing and modifying protocols for generating functional induced platelets (iPlatelets) from hPSCs. To reach high efficiency production and eliminate the exogenous antigens, media supplements and matrix have been optimized. In addition, the introduction of some critical transgenes, such as c-MYC, BMI1, and BCL-XL, can also significantly increase hPSC-derived platelet production; however, this may pose some safety concerns. Furthermore, many novel culture systems have been developed to scale up the production of iPlatelets, including 2D flow systems, 3D rotary systems, and vertical reciprocal motion liquid culture bioreactors. The development of new gene-editing techniques, such as CRISPR/Cas9, can be used to solve allogeneic immunity of platelet transfusions by knocking out the expression of B2M. Additionally, the functions of iPlatelets were also evaluated from multiple aspects, including but not limited to morphology, structure, cytoskeletal organization, granule content, DNA content, and gene expression. Although the production and functions of iPlatelets are close to meeting clinical application requirements in both quantity and quality, there is still a long way to go for their large-scale production and clinical application. Here, we summarize the diverse methods of platelet production and update the progresses of iPlatelets. Furthermore, we highlight recent advances in our understanding of key transcription factors or molecules that determine the platelet differentiation direction.
In mammals, platelets are produced by mature megakaryocytes (MKs) in the bone marrow and differentiate from pluripotent stem cells in hematopoietic tissues. The primary function of platelets is coagulation and hemostasis; once blood vessel injury occurs, platelets are rapidly activated, adhere to the wound, and aggregate to form a platelet clot. As a result, they are known as the “band-aids” of the bloodstream. Platelets play an executive role in the clinical treatment of blood diseases, such as acute myeloid leukemia, immune thrombocytopenia, and idiopathic thrombocytopenic purpura . Platelets are overlooked immune regulators; they play significant roles in inflammation and infection  as they can recognize exterior pathogens and produce many chemoattractants to activate and recruit leukocytes into the site of infection and inflammation, thereby enhancing their lethality to pathogens .
The roles of platelets in assisting liver regeneration, bone regeneration, and in the treatment of dermatological conditions, have also increased the demand for platelets in clinical treatment [4–6]. The discovery of platelet-derived serotonin involved in hepatic regeneration and the correlation between impaired platelets and liver cell proliferation suggest that platelets play a significant role in liver regeneration [7, 8]. Platelet transfusion can improve CCl4-induced liver fibrosis in mice with severe combined immune deficiency . The transfer of coding and regulatory RNA between platelets and hepatocytes can promote hepatocyte proliferation and liver regeneration [10–12]. After hepatectomy, platelets coordinate with liver sinusoidal endothelial cells and Kupffer cells via the release of various growth factors, including human growth factor, insulin-like growth factor, and vascular endothelial growth factor (VEGF), or through direct contact with hepatocytes [13–15]. As the therapeutic role of platelets in many diseases is being studied, the application of platelet-rich plasma (PRP) products has gained extensive attention in regenerative medicine. PRP is an autologous biological product derived from centrifuging or apheresis of blood and is a solution with high concentration of platelets [16, 17]. PRP treatment utilizes platelets with abundant biological factors and chemoattractive cytokines associated with tissue regeneration and remodeling.
Moreover, the hydrogel properties of activated PRP make it a suitable medicine delivery vehicle [7, 8, 18]. Platelets dynamically regulate the process of bone remodeling by releasing proinflammatory cytokines to activate the inflammatory phase of early bone healing and then enhance the repair phase of the healing process [19, 20]. PRP treatment has been widely studied in orthopedic and oral/maxillofacial injuries to aid hemostasis and musculoskeletal regeneration [5, 18, 21, 22]. Moreover, in aesthetic dermatology, PRP has been reported to have a therapeutic effect in treating hair loss caused by androgenetic alopecia . Combining platelets with fractional laser or fat grafting can improve scar revision [24, 25] and may provide benefits in skin rejuvenation and dermal augmentation [26, 27]. Thus, platelet therapy is expected to be a new therapeutic avenue for regenerative medicine and tissue engineering.
Previously, donor-derived platelets were the primary platelet source for the treatment of certain clinical diseases such as idiopathic thrombocytopenic purpura (ITP). However, the insufficient supply of donor blood limits its application worldwide. The complexity and doubts surrounding platelet donation have discouraged many donors, and current blood supplies do not meet clinical needs, causing severe shortages . In addition to this problem, there are also several inevitable challenges in platelet transfusion. The first is platelet preservation; platelets can only be stored at room temperature for a short time; otherwise, there is a significant risk of bacterial contamination. Although cold storage can reduce bacterial reproduction and prolong shelf life of the platelets, it also changes platelet structure, molecules, and metabolism . Second, exogenous platelets may cause excessive immune rejection in platelet recipients. Frequent platelet transfusions will cause allogeneic immunity, which results from the generation of multiple antibodies, such as human leukocyte antigen (HLA) antibodies and human platelet antigen antibodies in patients. Residual red blood cells (RBCs) in platelets can also induce RBC antibody production after transfusion . Exploring safe and high-quality alternative sources of platelets for clinical use will markedly benefit the field of regenerative medicine.
Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), which have the advantages of unlimited self-renewal and multiple directional differentiation capabilities, have become reliable platelet sources in regenerative medicine. Numerous studies have demonstrated that iPSCs can differentiate into various functional cell types, such as cardiomyocytes, nephron progenitor cells, kidney organoids, oligodendrocyte progenitor cells, and melanocytes [31–34]. Systems for generating induced platelets (iPlatelets) from human PSCs (hPSCs) have also been established using various methods [35–38]. Using gene-editing techniques, such as CRISPR/CAS9, PSCs with great genetic maneuverability can be developed; this makes PSCs more convenient and useful for overcoming some difficulties currently encountered by the use of platelets, such as allogeneic immunity. Therefore, hPSC-derived iPlatelets can overcome the limitations in the current blood donor-dependent system and solve a series of problems in platelet production for clinical application in the near future. However, there are still many challenges to overcome.
This review summarizes current approaches for generating hPSC-derived iPlatelets, presents the current status, compares the advantages and disadvantages, limitations, and defects, and suggests future research direction.
2. The Progress and the Current Approaches for iPlatelets
Many previous studies have reported that MKs are an essential intermediate product during hPSC differentiation into platelets, providing a new perspective for research and blood transfusion medicine. These studies are listed in Table 1; they describe MK differentiation and platelet generation in vitro (Figure 1).
VEGF, TPO, SCF, heparin
According to stage
BMP4, VEGF, SCF, TPO
BMP4, SCF, VEGF, FGF2
HUVECs (2D bioreactor)
CD41a⁺ or CD42b⁺
Higher than static condition
HPC, HEC, MKP, MK
About 30 platelets/MK
BMI1, BCL-XL, c-MYC
GATA1, FLI1, TAL1
About 7 platelets/MK
Shear stress (3D bioreactor)
~42 platelets/MK, ~350 platelets/h
2014  2016 
SCF, TPO, IL-9, IL-6
Turbulent flow, shear stress
HSC, HPC, MK
hESCs: human embryonic stem cells; hPSCs: human pluripotent stem cells; hiPSCs: human induced pluripotent stem cells; MK: megakaryocyte; HPC: hematopoietic progenitor cell; HEC: hematopoietic endothelial cell; MKP: megakaryocyte progenitor; HSC: hematopoietic stem cell; imMKCL: immortalized megakaryocyte progenitor cell line; TPO: thrombopoietin; VEGF: vascular endothelial growth factor; BMP4: bone morphogenetic protein 4.