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Patient Specific Stem Cell-derived Platelets - Coming to a Clinic Near You?

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Review of “Efficient Generation of Megakaryocytes From Human Induced Pluripotent Stem Cells Using Food and Drug Administration-Approved Pharmacological Reagents” from Stem Cells TM by Stuart P. Atkinson

Platelets (or thrombocytes) control the process of blood clotting and transfusions are common to prevent bleeding in treatments involving hematopoietic stem cell (HSC) and bone marrow (BM) transplantation [1]. Platelets are produced by mature megakaryocytes (MKs) in the bone marrow [2], and the production of both these types of cells in large quantities ex vivo would be highly clinically relevant. Differentiation of human pluripotent stem cells (embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs)) may be a useful strategy to create the supply to meet the high demand, although this currently remains inefficient or includes the use of xenogeneic and undefined reagents. To solve this problem, researchers from the groups of Zack Z. Wang (Johns Hopkins) and Linzhao Cheng (Johns Hopkins) have sought to create a robust and efficient means to generate expandable MK progenitors from hiPSCs using Food and Drug Administration (FDA)-approved pharmacological agents [3]. 

The group started by employing a modified 14 day two-step differentiation process to yield megakaryocytic precursors [4]. This process (see figure) started with spin-embryoid body (spin-EB) formation from cultures of single-cell hiPSC suspensions in 96 well plates, and the subsequent addition of BMP4 and FGF-2 to induce mesoderm differentiation, and then VEGF and SCF to induce hematopoietic differentiation. Thrombopoietin (TPO) addition at day 11 aimed to boost hematopoietic progenitor cell (HPC) formation and megakaryocytic lineage commitment, and by day 14, 30% of the hematopoietic cells released into suspension from the EBs expressed multiple megakaryocytic markers. The second step boosted MK differentiation by harvesting suspension cells and treating them with SCF, IL-11, and TPO for 5 days in 6 well plates leading to the enhancement on the number of mature large-sized MK-like cells. These cells produced platelet-like particles (PLPs) similar to those isolated from human peripheral blood following an additional 18 days of culture in a chemically defined media without feeder cells.

While this strategy represents an efficient strategy for the production of MKs, adaption for clinical applications requires the elimination of all animal products and the use of clinical grade reagent. To comply with this necessity, the group employed the FDA-approved TPO analog romiplostim (Nplate), and replaced BSA with in serum-free medium with Plasbumin, which is a clinic-grade agent enriched for HuA made from serum plasma. Encouragingly, these replacements, when used alongside either recombinant human proteins or human-sourced proteins, further enhanced MK production, allowing for a robust and consistent feeder and xeno-free system when assessed from 44 additional hiPSC lines derived from 22 individuals.

While the authors do note that further tweaks to the process may be required to remedy the relatively low amount of PLPs produced, this still represent an excellent first step towards the clinical applicability of this therapeutically relevant cell type. The authors also highlight a recent report on the production of platelet from mouse fetal-liver culture-derived MKs using a biomimetic microfluidic platelet bioreactor [5], which may allow the further enhancement of MK production from hiPSCs by the recapitulation of the bone marrow and blood vessel microenvironments.

References

  1. Stroncek DF and Rebulla P Platelet transfusions. Lancet 2007;370:427-438.
  2. Shultz LD, Lyons BL, Burzenski LM, et al. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells. Journal of immunology 2005;174:6477-6489.
  3. Liu Y, Wang Y, Gao Y, et al. Efficient generation of megakaryocytes from human induced pluripotent stem cells using food and drug administration-approved pharmacological reagents. Stem Cells Translational Medicine 2015;4:309-319.
  4. Ng ES, Davis RP, Azzola L, et al. Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation. Blood 2005;106:1601-1603.
  5. Thon JN, Mazutis L, Wu S, et al. Platelet bioreactor-on-a-chip. Blood 2014;124:1857-1867.