Page 36 - 2021_04-Haematologica-web

C.A. Di Buduo et al.
feeder cells.65-67 Other strategies include the use of sero- tonin, which supports megakaryocyte maturation and proplatelet formation by activating biochemical pathways and through modulation of cytoskeleton dynamics.70 The cytoskeleton is responsible for controlling membrane stiff- ness and resistance to deformation of megakaryocytes induced by environmental pressures. Inhibition of the actomyosin cytoskeleton by blebbistatin and Rho/ROCK inhibitors can soften the membrane and facilitate the frag- mentation of megakaryocytes.71,72 However, given the importance of cytoskeleton remodeling during the whole process of differentiation, such treatments should be administrated only to mature megakaryocytes at the stage of proplatelet formation. StemRegenin1, an antagonist of the aryl hydrocarbon receptor, was shown to specifically increase the expansion of CD34+CD41low early megakary- ocytic progenitors and to promote the capacity to generate proplatelets and platelet-like elements.73 Subsequently, a high throughput screening by Seo et al. identified new inducers of megakaryocyte maturation and platelet pro- duction, such as CH223191, Wnt-C59 and TCS359, respectively inhibitors of the aryl hydrocarbon receptor and of the Wnt and Fms-like tyrosine kinase 3 pathways.74 However, studies on their mechanisms of action are need- ed before a conceivable application for clinical purposes.
Most of the cited compounds have been studied in liq- uid cultures that lack the shear forces of blood flow to sup- port the elongation process and platelet release. To face this challenge, microfluidic chips with flow chambers and fenestrated barriers functionalized with extracellular matrix components have been proposed.75-77 Thon et al. established the first microfluidic chip. The device was made of transparent silicon and supported high-resolution live-cell microscopy and quantification of platelet produc- tion. It consisted of upper and lower microfluidic channels separated by a 2 mm fenestrated barrier. Megakaryocytes were seeded in the upper channel and extended pro- platelets through the slits.75 Later on, aiming to increase the yield of collected platelets, Avanzi et al. created an innovative bioreactor made of a pseudo-3D membrane, either a nanofiber membrane or a polyvinyl chloride filter, placed between two 3D-printed flow chambers. The upper side of the membrane housed megakaryocytes, and the lower compartment was a flow chamber destined to harvest platelets.76 Blin et al. designed an evolution of these chips.77 Their microfluidic device consisted of a microchannel textured with organized micropillar arrays coated with von Willebrand factor to anchor megakary- ocytes while promoting platelet rolling into the flow. All these systems were able to provide a hydrodynamic shear supporting proplatelet elongation and fragmentation into platelets, but still with low efficiency in terms of numbers for clinical application because of their micro-scale nature.
Two- to four-phase cultures using combinations of ery- thropoietin and various growth factors, steroids and cytokines, with or without serum and/or feeder layers have been developed to reproduce complete erythro- poiesis. A multistep process is needed to control the fine balance between cell expansion, differentiation and matu- ration. Cell expansion at the stage of stem cell progenitors has been carried out in the presence of stem cell factor, thrombopoietin and/or Fms-like tyrosine kinase 3.78 Delta 1 Notch ligand increased the proliferation rate of early progenitors but the differentiation process was delayed in this culture.79 Insulin-like growth factor 1 has been used to
promote stem cell survival and to guide erythroid differen- tiation, and it has been validated as a promoter of nuclear condensation but not of enucleation.80,81 Erythroblast enu- cleation is thought to be largely dependent on signals mediated by macrophages, but mimicking erythroblastic islands is challenging and different approaches have been proposed to enhance its occurrence in vitro. The basic method consists of seeding erythroid cells with feeder cells, such as murine and human stromal cells or human monocyte-derived macrophages, but differing effects on maturation and enucleation have been reported.49,82,83 Recently, Lopez-Yrigoyen et al. established genetically programmed hiPSC-derived macrophages to develop an elegant approach of co-culture with umbilical cord blood- and hiPSC-derived erythroid cells that demonstrated effi- cient maturation and enucleation,84 although the presence of feeder cells could make it difficult to isolate pure, non- contaminated erythrocytes. A second approach is to inject erythroid precursors into immunodeficient mice in order for the cells to complete their maturation, but this is clear- ly not applicable for obtaining cells for clinical purposes.52 In a third approach different protocols have been devel- oped to produce enucleated erythroblasts in the absence of feeder cells, including culture with a cytoprotective polymer called poloxamer 188 which increases membrane stability during the enucleation process.85 The fourth approach involves seeding cells in agitated bioreactors sys- tems. Timmins et al. used a commercially available device consisting of a rocking platform that guaranteed homoge- neous mixing with low shear.86 In this system feeder cells were not essential for proficient expansion and terminal differentiation of erythrocytes. The same results were obtained with a bottle-turning device culture system87 and
88
more recently with a stirred‐tank bioreactor. Culture in
suspension is an approach that can be used for large-scale production of blood cells, including megakaryocytes. Constant agitation during culture, generated by a rotary cell culture system and stirred spinner flasks, demonstrat- ed the ability to produce platelets.89 More recently, the group of Koji Eto used big tank bioreactors to develop a scalable, controllable, turbulent flow-based system for platelet generation.18 The breakthrough of the big bioreac- tors was their ability to guarantee appropriate oxygena- tion and prevent cell conglutination while providing dynamic flows mimicking those of the bloodstream.
In most of these conditions erythrocytes and platelets look immature; in particular, erythrocytes appear macrocyt- ic with a large amount of fetal hemoglobin and platelets appear larger with immature granules. It is known that cul- tured megakaryocytes of any origin produce fewer platelets per single cell than do platelets in vivo. We can hypothesize that this is because they miss the physical environment of the bone marrow which drives maturation of HSC and the final production of erythrocytes and platelets in vivo. Thus, one of the major challenges in this field of research is to implement culture protocols with systems able to provide the most favorable conditions for mimicking the bone mar- row hematopoietic niche.
The rise and perspectives of the three-dimensional bone marrow mimic
In the last decades, mechano-biological studies have consolidated the importance of the physical environment,
952
haematologica | 2021; 106(4)