What are platelets?
- second most numerous cell in the body
- produced from megakayrocytes
- anucleate and highly granular
- when activated, take on a filapodial form
What are some processes where platelets are involved?
- haemostasis (stopping bleeding)
- thrombosis
- metastasis
- sepsis
- inflammation
- many roles rely on granule content
Describe platelet granules
two types
- alpha granules: larger and more abundant. contain (anti)coagulants, membrane proteins, growth factors, ce=hemokines and immune and adhesion factors
- dense granules: contain cations, nucleotides, ATP, bioactive amines
How do platelets stop bleeding?
constantly surveilling the endothelium. sense damage by surface receptors and activate their granule contents. recruit other platelets and trigger a coagulation cascade resulting in the formation of a haematopoetic plug that stops the bleeding
What are megakaryocytes?
- platelet precursors made in the bone marrow
- common myeloid progenitors
- multinucleated and large
- endomitosis - gets prepared to divide but never does so DNA increases and cell size increases
- thrombopoietin cytokine drives differentiation
How do megakaryocytes become platelets?
- not fully understood
- mature MKs migrate to perivascular environment
- inhibit the ECM, remodel the cytoplasm + membrane to squeeze into blood vessels
- elongated proplatelets, large proteins and whole MKs enter the vessels
- broken down into platelets at the lung vasculature due to interactions with endothelial cells and blood flow stress
What are some typical uses of platelets in the clinic?
- prevent bleeding in thrombocytopenia
- in response to cancer treatment leading to bone marrow malignancy
- in surgery or in response to haemorrage
- congenital (Glanzmanns) and aquired (NSAID) platelet defects
What are the two major clinical sources of platelets currently?
- spun from whole blood donations with leukocytes removed by filtration
- collected from a donor by apheresis - platelets are separated and the rest of the blood is returned to the circulation of the donor
Why is there a need to make platelets in vitro?
- increased demand with an aging population
- reduced supply and a short shelf life
- infection risk from donors
- immune mismatches
What are the two main challenges with making platelets in vitro?
- mechanisms underlying megakarypoesis not fully understood
- would need 75-150 million mature MKs to make the equivalent of one pack of donated platelets
What is the general process of creating platelets in vitro?
- take human stem cells and differentiate
- or human adipocytes/fibroblasts + transdifferentiate in 2D culture
- add to bioreactor to induced platelet formation
- blood type matching and use
What are the positives and negatives of using haematopoetic stem cells to make platlets in vitro?
- bone marrow + peripheral blood HSCs differentiate well into MKs
- rare in the circulation
- multipotent
- can’t renew forever in culture
- asynchronous cultures
What are the positives and negatives of using iPSCs to make platelets in vitro?
- self renewing
- can genetically define and modify
- proliferation and differentiation can be controlled to synchronize maturation
- make lower platelet / MK yields
- mature less well
What is asynchronous culture of stem cells?
- can’t control when each cell matures
- many cells at different stages
How can iPSCs be used to create immortalised platelet progenitors?
- introduce c-myc, BMI-4, BCL-XL to iPSC-derivedMK precursors or HSCs
- induce constant proliferation
- The addition of doxycyclin to MKs causes a huge expansion of MKs
- when dox is turned off, platelet production begins and all cells one in sync
What 3 factor are important to consider when designing bioreactors for platelet production?
- wall shear stress as blood moves along the endothelium
- difference in flow from the middle to the outer edges of vessels
- Reynolds number - predicts flow conditions more smooth or turbulent?
What are some current bioreactor examples for platelet production?
- bioreactors with many narrowing chambers
- flow pushes MKs through
- mimics lung vasculature
- forces palatlets to form
What are some potential furutre uses of stem cell derived platelets?
- could genetically modify to activate at specific sites
- could load granules with helpful contents to treat disease or drive expression of desired proteins
