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C.A. Di Buduo et al.
concerns regarding potential transmission of prions and viruses.7,8 The recent spread of Sars-CoV-2 has had a pro- found impact on the number of blood donations, blood component supplies, and safety.9,10 The Sars-CoV-2 has a long incubation period and causes asymptomatic infec- tions in most people, which poses enormous challenges in the recruitment of blood donors, necessitating the imple- mentation of new screening guidelines for hemovigilance. Indeed, during the lockdown, blood transfusion centers experienced a dramatic drop in the number of volunteers almost worldwide. Furthermore, several agents first described decades ago still represent ongoing blood safety risks that have not been adequately addressed. These include several species of Babesia known to cause human infections, which are being reported more frequently every year, especially in the USA.11
Given all this background, ex vivo manufacture of ery- throcytes and platelets is becoming an increasingly attrac- tive approach for transfusion medicine. In this review, we discuss the most recent scientific knowledge about mech- anisms of erythrocyte and platelet production and techno- logical advances in the field of bioengineering, which together have led to the development of new laboratory tools that mimic human bone marrow with different lev- els of complexity. The breakthrough of these approaches will lead to the generation of highly defined and con- trolled microenvironments in a clinical-grade condition for producing blood cell units on demand for transfusions.
Looking inside the bone marrow: an overview
on the origin of blood platelets and erythrocytes in vivo
The formation of blood cells from hematopoietic stem
cells (HSC) occurs within the bone marrow through a
series of ever more differentiated progenitors under the
tight control of soluble and environmental factors that
cooperate in a framework known as the hematopoietic
niche.12 Within this context, millions of platelets and ery-
throcytes are generated each day from a common
megakaryocyte-erythroid progenitor cell that is recruited
towards final differentiation by thrombopoietin or ery-
thropoietin (Figure 1).13 A subset of long-term HSC with
restricted myeloid-repopulating activity committed to the
erythro-megakaryocytic lineage has also been postulat- ed.14,15
During their process of differentiation, both cell lineages undergo characteristic morphological changes that lead to the respective lineage consolidation. These include nuclear polyploidization and cellular enlargement with the development of cytoplasmic granules and the demar- cation membrane system for megakaryocytes,16 while cell size reduction and chromatin condensation accompanied by increased production of hemoglobin can be observed in erythroblasts.17 Both cell lineages face one crucial final step at the end of their maturation. For megakaryocytes this is elongation of thin pseudopods, known as proplatelets, within the lumen of bone marrow sinusoids, where platelet detachment from the proplatelet shaft can be attributed mainly to turbulent blood hydrodynamics and fluid shear.18 A recent study demonstrated that membrane budding also contributes to supply the platelet biomass.19 For erythrocytes the crucial step entails expulsion of the nucleus from erythroblasts, which leads to the formation
of reticulocytes, and the loss of organelles and ribosomes through autophagy/exosome-combined pathways.20
While cytokine-mediated priming is important to drive hematopoietic cell commitment, the final steps are strictly dependent on the interplay of different environmental cues, including cell-to-matrix and cell-to-cell interac- tions.21,22 Both platelet and erythrocyte production are pro- foundly influenced by the composition and stiffness of the extracellular matrix, which can model cell behavior through mechanical and chemical signals via integrins and mechano-sensitive ion channels.23-25 The protein tangle shaping the extracellular matrix is mainly composed of different types of collagen (I, II, III, V, XI), fibronectin, laminins, and glycosaminoglycans, continuously remod- eled in physiology and disease through specific proteases, such as matrix metalloproteinases.26 It has been demon- strated that the softest substrates of the extracellular matrix, such as fibronectin and type IV collagen, mainly located in the medullary cavity and around sinusoids, support megakaryocyte proliferation and proplatelet for- mation. In contrast, the endosteal surface, which is asym- metrically enriched with the stiff type I collagen, prevents platelet production.27,28 Regarding erythropoiesis, the interaction with fibronectin also supports cell prolifera- tion and protects from apoptosis.29 Specialized niches localized throughout the intratrabecular space support erythrocyte maturation. Early erythroid progenitors are closely associated with perisinusoidal leptin receptor-pos- itive stromal cells that secrete stem cell factor to support their maintenance,30 while the process of enucleation takes place within erythroblastic islands, hematopoietic sub-compartments composed of erythroblasts surround- ing a central macrophage.31,32 Here, the interaction between macrophages and erythroblasts, mediated by the erythroblast-macrophage protein and integrins, is required to facilitate proliferation and differentiation and provide iron to the erythroblasts.33
In the light of all this knowledge, the top three chal- lenges facing biomedical research today aimed at develop- ing clinically relevant tools for ex vivo blood cell produc- tion are: (i) finding appropriate sources of stem cells; (ii) identifying efficient culture conditions for their commit- ment; (iii) mimicking relevant features of the bone mar- row microenvironment to support the final stages of ery- throcyte and platelet production.
The artificial cell and stem cell pipelines for obtaining functional platelets and erythrocytes in vitro
Artificial blood cell production and hematopoietic induction of stem cells have been studied to generate in vitro fully functional platelets and erythrocytes (Figure 2).
Synthetic platelets able to adhere to subendothelial structures have been constructed by functionalizing poly- meric, liposomal or discoid albumin particles with recom- binant glycoproteins or small peptides that bind to von Willebrand factor and collagen.34 More recent advances in the field include: highly deformable microgel platelet-like particles, which tenaciously bind to fibrin fibers promot- ing clot contraction and stability;35 platelet-like nanoparti- cles, whose discoidal shape and flexible exterior enhance platelet marginalization and aggregation;36 and artificial dense granules consisting of liposomes that release factors
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haematologica | 2021; 106(4)