Baffled‐flow culture system enables the mass production of megakaryocytes from human embryonic stem cells by enhancing mitochondrial function

Abstract

Human embryonic stem cells (hESCs) have become an ideal cell source for the ex vivo generation of megakaryocyte (MK) and platelet products for clinical applications. However, an ongoing challenge is to establish scalable culture systems to maximize the yield of stem cell‐derived MKs that release platelets. We defined a specific dynamic 3D manufacturing system in a baffled‐flow manner that could remarkably facilitate megakaryopoiesis and increase the yield of platelet‐producing MKs from hESCs within a 12‐day induction period. Additionally, an increased number of >16N ploidy MKs, proplatelets, and platelets were generated from induced cells harvested on Day 12 using the specific dynamic culture method. The specific dynamic culture method significantly enhanced endothelium‐to‐haematopoietic transition and early haematopoiesis. More importantly, MK fate was significantly facilitated in a specific dynamic manner during early haematopoiesis. Mechanistically, this dynamic culture significantly enhanced mitochondrial function via the oxidative phosphorylation pathway and caused differentiation skewing of hESCs toward megakaryopoiesis. This study can aid in the automatic and scalable production of MKs from stem cells using baffled‐flow bioreactors and assist in the manufacturing of hESC‐derived MK and platelet products.

Establishment of human hematopoietic organoids for evaluation of hematopoietic injury and regeneration effect

“…1 b, c). Subsequently, populations of hematopoietic-like cells were selectively enriched using CD34 + immunomagnetic bead sorting [ 21 ]. Flow cytometry analysis revealed high expression levels of CD43, CD34, and CD45 in these cells, with CD43 + CD34 + cells comprising approximately 93.70% and CD34 + CD45 + cells constituting approximately 38.50% of the population (Fig.…”

“…Afterwards, cells were rinsed with phosphate-buffered saline (PBS) and transitioned to Stage II BEL medium, enhanced with 50 ng/ml VEGF (R&D Systems), 20 ng/ml bFGF (Selleck), and 2 µM SB431542 (Selleck), and incubated for 72 h. After incubation, the cells were dissociated into single-cell suspensions. CD34 + cells were then isolated by positive selection using a MACS CD34 MicroBead Kit (Miltenyi Biotec, Gladbach Bergisch, Germany) [ 19 – 21 ]. Isolated CD34 + cells were then resuspended in BEL medium containing 50 ng/mL SCF (PeproTech), 20 ng/mL TPO (PeproTech), 20 ng/mL IL-3 (PeproTech), 20 ng/mL Flt3L (PeproTech), 20 ng/ml VEGF, 10 ng/mL bFGF, and 5 µM SB431542.…”

“…A 100 μL cell suspension was then combined with 0.5 mL of H4636 hematopoietic colony culture medium (STEMCELL Technologies), thoroughly mixed, and dispensed into ultra-low attachment 24-well plates. After 14 days of incubation, colonies were enumerated and classified based on morphology into erythroid (BFU-E, burst-forming unit-erythroid), myeloid (CFU-GM, colony-forming unit-granulocyte, monocyte), or mixed lineage (CFU-GEMM, colony-forming unit granulocytes, erythrocytes, monocytes, and megakaryocytes) categories [ 21 ].…”