Bone marrow niche-inspired, multiphase expansion of megakaryocytic progenitors with high polyploidization potential

Abstract

Background-Megakaryopoiesis encompasses hematopoietic stem and progenitor cell (HSPC) commitment to the megakaryocytic cell (Mk) lineage, expansion of Mk progenitors and mature Mks, polyploidization, and platelet release. pH and pO 2 increase from the endosteum to sinuses, and different cytokines are important for various stages of differentiation. We hypothesized that mimicking the changing conditions during Mk differentiation in the bone marrow would facilitate expansion of progenitors that could generate many high-ploidy Mks.

Three-StageEx VivoExpansion of High-Ploidy Megakaryocytic Cells: Toward Large-Scale Platelet Production

“…Our previous work focused on the production of Mk progenitors with the potential to yield high-ploidy ( > 4N) Mks. 29 Besides identifying an effective cytokine combination, we showed that shifting the Mk progenitors from 5% O 2 and pH 7.2 to 20% O 2 and pH 7.4 at day 5 greatly increased mature Mk production.…”

“…[26][27][28] We recently achieved a yield of 14 Mks per mPB HSPC and obtained up to 45% highploidy ( > 4N) Mks. 29 This Mk productivity has the potential to allow production of one platelet transfusion from 125 million mPB CD34 + cells, but it is clear that the yield must be increased by another order of magnitude.…”

Platelet bioreactor-on-a-chip

“…Mounting evidence that cell-cell contacts, 22,23 ECM composition 19,20 and stiffness, 21 vascular shear stress, 24,25 pO2/pH, 51,52 soluble factor interactions, 22,53 and temperature 54 contribute to proPLT formation and PLT release have suggested that recapitulating key components of BM and blood vessel microenvironments within a three-dimensional microfluidic culture system is necessary to achieve clinically significant numbers of functional human PLTs. Our microfluidic bioreactor design expands control and resolution of the microenvironment, and has allowed us to drastically improve the time to PLT release (from 18 hours down to 2 hours) and more For personal use only.…”

Challenges and promises for the development of donor-independent platelet transfusions

“…Perhaps large-scale, continuous harvesting techniques using bioreactor technologies, outside the scope of most research laboratories, are needed to address this challenge. In the meantime, several groups have begun exploring replication of the physical nature of the bone marrow niche, including altering the physical rigidity of the culture environment, 35 decreasing oxygen tension, 36 increasing culture temperature, 37 or simulating blood flow shear conditions 38 to enhance platelet yield. Shin et al 35 hypothesized that a soft matrix for cell culture as well as sustained inhibition of nonmuscle myosin-II would maximize megakaryocyte maturation and platelet release.…”

“…Incorporation of low oxygen tension during the early phases of megakaryopoiesis also increased the numbers of mature megakaryocytes. 36 To simulate a complex marrow niche, a woven polyester surgical fabric in a 3-dimensional single-pass perfusion bioreactor system was developed, which allowed the continuous collection of platelets for over 30 days with yields of up to 36 3 10 6 platelets per day per bioreactor. 40 Subsequently, the same group was able to start with ;2 3 10 6 CD34 1 cells, vary oxygen tension in this system, and produce approximately 300 platelets per starting cell over 30 days.…”