Platelet production: Developments and Challenges
Platelets are anucleate cells circulating in peripheral blood, which form a multicellular plug at the site of injury. In a way they are nature’s “band-aids”, which preventing excess blood loss. Life of a platelet begins in the form a hematopoietic stem cell (HSC) in the bone marrow. An intricate interplay of transcriptional networks along with a combination of cytokines, enable the HSCs to commit to the megakaryocyte (MK) lineage. MKs are giant, polyploid (multi-chromosomal) cells which develop in the bone marrow and serve as platelet precursors. They migrate towards the vascular sinusoids in the bone marrow and extend finger-like protrusions called proplatelets into the circulating blood in the blood vessels. Shear forces of circulating blood break down the proplatelet extensions in platelets [Figure-1].
Hence it is not surprising that fall in circulating platelets levels (thrombocytopenia) results in clotting disorders and excess blood loss. Thrombocytopenia is frequently reported in individuals with bone marrow failure, cancer patients receiving chemotherapy, bone marrow grafts and can also result from autoimmune issues and side effects of certain drugs. Transfusion of donor platelets is only treatment available for these conditions. However, the platelets shelf life is low, often leading to unavailability of adequate quantities. Indeed, USA has seen a 7.3% rise in platelets transfusions between 2008 to 2011. Furthermore, other complications include possibility of bacterial contamination, alloimmunization and platelets refractoriness. To overcome these shortcomings - generation of platelets in-vitro, is the need of the hour.
However, generation of adequate quantities of functional platelets in-vitro, is challenging. The major limiting factors include:
Generation of sufficiently high quantities of MKs in culture which will be subjected to platelets biogenesis. Moreover, the overall morphology of the cytosolic structures like demarcation membrane system (DMS) and ploidy levels of in-vitro generated MKs should closely mimic the MKs developing in-vivo in the bone marrow.
Design mechanisms for efficiently breaking down the proplatelets to ensure generation of substantial quantities of platelets in-vitro
To ensure that the transfusion of in-vitro generated platelets will result in successful hemostasis.
How to ensure higher platelets yields? :
Choice of progenitors for platelets engineering in-vitro
The source of cells i.e., iPSC or hESC is an important factor determining the yield of substantial quantities of differentiated MKs. Although both these cell types are pluripotent, the iPSCs have can override the ethical concerns associated with iPSCs. In many cases used of CD34+ progenitors isolated from peripheral blood, bone marrow or cord blood can be advantageous because the source materials can be handled easily, overall efficiency of platelets yield and close resemblance of platelets ultrastructure to platelets generated in-vivo. However, a major drawback of CD34+ cells lies in their limited expansion potential. However, Nakamura et al., have generated an expandable MK line capable of giving rise to platelets in-vitro by overexpressing c-MYC and BCLXL. Another such immortalized MK line has been developed by Moreau et al., by overexpressing transcription factors such as FLI1, GATA1 and TAL1. Both these lines have been reported to generate platelets with higher efficiency and in shorter time frames compared to iPSCs. Furthermore, the advent of CRISPR-Cas9 genome editing technology has been an additional boon for generating HLA-1 deletions in MKs and platelets, which can overcome platelets refractoriness after transfusion. In-spite of the many promising advances in the molecular genetic techniques, a major challenge remains the ability to generate industrial scale production of artificial platelets to keep up with the demands.
Enable adequate maturation of MKs in ex-vivo conditions
To ensure generation of sufficient quantities of platelets from in-vitro cultured MKs, it is essential to maintain high ploidy levels in these precursor cells, since smaller MKs with lower ploidy generate fewer platelets. Pharmacological perturbation of actomyosin signaling components can regulate ploidy levels. Recently, Elagib et al., have also reported the downregulation of an RNA binding protein leads to increase in ploidy and cell size transforming fetal MKs to develop more adult MK like features.
Another cytoskeletal structure, which is a crucial determinant of platelets generation efficiency of MKs is the DMS. The DMS is a reticular, inner membrane system in MKs which serves as a membrane reservoir for the proplatelets emerging from the MKs. Recently, Aguilar et al, have demonstrated the importance of stiffness of the different bone marrow niches in regulating DMS architecture. Furthermore, lipid biosynthesis and uptake by immature MKs regulate DMS maturation. Thus, a clear understanding of these metabolic pathways can reveal important insights into MK maturation.
Ensuring interaction of MKs with bone marrow niches
Another impediment in generating in-vitro platelets production with high yield lies in the inability to recreate the MK-endothelial interaction in an artificial setting. MKs migrate to the blood vessels in the bone marrow and extend proplatelets, through the endothelial cells lining the vasculature. Specialized adhesive structures in the MKs called the podosomes release proteolytic enzymes, which degrade the extracellular matrices allowing the proplatelets to enter the vascular lumen. Antkowiac et al., has demonstrated that endothelial cells lining the sinusoids enhance podosome formation and subsequently lead to DMS polarization prior to proplatelets generation. Hence, bioreactors with endothelial cell containing 3D flow system need to be designed to increase platelets yields. Furthermore, bioreactors and flow systems should be appropriately designed to mimic the trajectory and vectorial parameters similar to circulating blood in bone marrow sinusoids.
Thus, although substantial progress has been made to greatly improve the ex-vivo generation of platelets, a number of concerns need to address in the future to enhance high platelets yields using artificial systems. This includes optimization and fine-tuning of current methods along with better insights into the cellular and molecular mechanisms of platelets biogenesis in physiological systems.