3D Model of Thrombopoiesis.
Authors: Pallotta Isabella , Lovett Michael L. , Kaplan David L. , Balduini Alessandra 
Published: October 2019

Abstract
Abstract 1609

Background.
The mechanisms that regulate megakaryocytic (Mk) development within the bone marrow environment remain poorly understood. The underlying relationships between Mk maturation and bone marrow components are key factors in this process. Mk development occurs in a complex microenvironment where extracellular matrices are fundamental regulatory components. The first events occur in the osteoblastic niche and include commitment of the hemopoietic progenitor cell to Mk, arrest of proliferation and initiation of endomitosis. The second step is Mk maturation and is associated with rapid cytoplasm expansion and intense synthesis of proteins. Finally Mks, which migrate to the vascular niche, convert the bulk of their cytoplasm into multiple long processes called proplatelets that protrude through the vascular endothelium into the sinusoid lumen, where the platelets are released.

Hypothesis.
The hypothesis for the present work is that a complex in vitro 3D bone marrow-like environment can be used to gain fundamental mechanistic insight into cell signalling and matrix-cell interactions in the bone marrow niche related to Mk development.

Methods.
We propose the first 3D model for Mk function in the bone marrow environment, by refining a recently proposed bioreactor platform (Lovett et al., 2007). These bioreactors consist of 3 wells (10 mm × 15 mm × 5 mm) within a PDMS block (25 mm × 60 mm × 5 mm) which is plasma bonded to cover glass for imaging. Each bioreactor well was perfused by 23 G stainless steel needles, spanned by porous silk microtubes as blood vessel scaffolds (640 μm inner diameter), positioned approximately 500–750 μm from the bottom of the bioreactor and connected to tubing for media perfusion using a programmable syringe pump. These microtubes were prepared by dipping several times straight lengths of stainless steel wire into 10–14% (w/v) aqueous silk fibroin to obtain blood vessel scaffolds with a wall thickness of around 50 mm. Defined pore sizes of 6–8 μm were obtained by adding 6 w/t % poly(ethylene oxide) (PEO) to the silk fibroin. The perfused silk tubes comprised the vascular niche and were embedded within a cell-seeded hydrogel which comprises the osteoblastic niche. The silk microtubes were coated with a combination of fibrinogen, von Willebrand Factor, type IV collagen and SDF-1 alpha, to better establish the composition of the vascular niche. Control experiments were performed by coating silk microtubes with type I collagen. After staining human umbilical cord blood derived Mks, the cell suspension was added to the hydrogel and Mk migration was analyzed in a time-dependent manner using confocal microscopy analysis. Further, flow effluent through the vascular tubes in the bioreactor was collected at regular time intervals and platelet numbers and function were analyzed by flow cytometry and microscopy. Culture released platelets were counted as CD61+ events with the same scatter properties of human blood platelets.

Results.
Our results showed that Mks migrated towards the vascular microtube coated with Fibrinogen, von Willebrand Factor, type IV collagen and SDF-1. Mks were also able to complete their maturation in the proximity of the microtube by extending proplatelets. Interestingly, confocal microscopy analysis revealed that Mks were able to extend proplatelets through the vascular microtube wall and release CD61+ platelet-like particles inside the vascular microtube. Cytofluorimentric analysis demonstrated that the particles collected in the flow effluent of the vascular microtube were CD61+ cells with the same scatter properties of human peripheral blood platelets. Finally, upon coating with only type I collagen Mks did not migrate towards the vascular microtube or extend proplatelets to release platelets. Thus, by mimicking the relationship between Mks and the bone marrow environment, a model to reproduce the different steps of Mk development, such as Mk migration, proplatelet formation and platelet release, is established. This is a first significant step towards relevant systems for the study of these cellular processes in detail as well as toward potentially useful in vitro platelet production systems.

Conclusions.
In this work we developed a new 3D bone marrow system in vitro that could represent a new tool to understand the mechanistic basis for Mk development and function, and the diseases related to these cells.

Disclosures:
No relevant conflicts of interest to declare.