In Vitro Production Of Megakaryocytes and Platelets From Human Induced Pluripotent Cells By GMP Compatible Methods

Human pluripotent stem cells and in particular induced pluripotent stem cells (iPSC) derived from adult tissue have recently opened novel and promising avenues for cell therapy. In vitro production of high demand transfusion products such as platelets from HLA- and HPA-typed iPSC lines is an attractive target but to achieve this potential there is a clear need for reliable and efficient GMP compliant differentiation protocols and dedicated cell culture devices compatible with large scale cell production.

We first developed a novel protocol for production of megakaryocytes (MKs) from human iPSCs based on the ectopic expression of specific transcription factors (TFs), so called “forward programming”. Through a transcriptome and protein interactome guided process, we initially shortlisted 46 TF candidates and eventually tested fourteen of the top ranked factors for their forward programming potential. Using lentiviral vectors we identified a minimal combination of three TFs which generated the highest amount of MKs. This forward programming protocol was able to robustly induce MK differentiation from various embryonic and iPSC lines. Further developments have lead to a fully standardise differentiation system using forced aggregation of defined amounts of single iPSCs into embryoid bodies and chemically defined media throughout the culture. We reliably achieve a 99% pure MK culture with a mature phenotype (i.e. CD41a+/CD61+/CD42a+/CD42b+/GP6+, polyploid and forming proplatelets and expressing key regulatory genes (e.g. NFE2, MEIS1, PBX1, RUNX1, ZFPM1) and a cell yield in excess of 50 times the initial iPSC input. These results surpass existing protocols offering wider perspectives in using human iPSC derived MKs for biological studies as well as clinical applications.

This field of research has also been hampered by the lack of efficient, GMP-compatible protocols to derive platelets from MKs. We set out to address this issue by creating a 3-dimensional biomimetic/biocompatible scaffold to be incorporated a perfusion bioreactor to mimic the chemico-mechanical signals to enhance proplatelet formation and platelet release from MKs and allow the harvest of functional platelets in numbers compatible with those required for human use.

The importance of the niche environment (including cell-to-cell contact and extracellular matrix proteins) for both MK maturation and platelet release has been demonstrated in different studies. Based on protein expression data from endothelial cells and mesenchymal cell lines that support platelet formation in co-cultures, we have generated a library of recombinant transmembrane proteins (TMPs). These TMPs contain the extracellular domain of the candidate transmembrane proteins at the N-terminal while at the C-terminus they contain a sequence which allows purification and immobilization on either culture plates or 3D scaffolds.

We have used a combinatorial approach to perform a high throughput screening of the TMP library identify which TMPs promote proplatelet formation. Briefly, TMPs were immobilized onto 96-well plates onto which mature culture-derived MKs were seeded and proplatelet formation recorded by microscopy and digital imaging analysis. The best candidate combinations were identified through a mathematical model adapted from drug screening experiments.

Finally we have produced porous matrices using GMP-grade collagen and fibrinogen via a freeze-drying process whereby ice crystals introduced into a protein-acid slurry are sublimated off to produce a fully interconnected porous structure. The structure can be tailored in terms of pore sizes and morphologies by varying the temperature profile. The structural integrity over prolonged periods in tissue culture can be enhanced by chemical crosslinking, which also removes the active moiety of collagen. We show that both collagen and fibrinogen scaffolds support MK culture from cord-blood derived CD34+ stem cells with no toxic effects and similar differentiation properties as with standard 2D cultures. We also show that the scaffold support platelet formation from seeded mature MKs. These inert scaffolds can be further functionalized by affixing the candidate TMPs identified above via a UV-light driven process onto the inert base scaffold to give a direct contact signal to the MKs to produce platelets.