Ex Vivo Induction of Megakaryocytic Differentiation of Umbilical Cord Blood CD34⁺ Cells

Background and Objectives: Due to platelet shortage, megakaryocytes have been regarded as an effective substitute to the alleviation of frequent thrombocytopenia after stem cell transplantation. However, ex vivo expansion of megakaryoblasts and their subsequent differentiation into mature megakaryocytes for clinical applications remains a challenge. Here, we describe the development of a two-stage culture system for producing megakaryocytes from cord blood CD34⁺ cells.

Design and Methods: Firstly, we expanded CD34⁺ hematopoietic progenitor/stem cells for 6 days in a serum-free culture system (IMDM basal medium with the addition of biotin, putrescine, insulin, human serum albumin, selenium, and some other nutrients) supplemented with stem cell factor (SCF), Flt-3 ligand (FL), thrombopoietin (TPO), interleukin 3 (IL-3), low density lipoprotein (LDL), StemRegenin 1 (SR1), and DMSO. CD34⁺ cells expansion was monitored by flow cytometric analysis of cell surface markers coupled with cell counting. Subsequently, these expanded cells were induced toward the megakaryocytic lineage for additional 7 days in the same serum-free medium as above supplemented with SCF, TPO, IL-3, IL-6, IL-11, granulocyte-macrophage colony-stimulating factor (GM-CSF), and LDL. Megakaryocytes were detected by flow cytometry using antibodies against specific cell surface markers including CD41a and CD42b. Differentiated megakaryocytes were also confirmed by morphological criteria such as cell size and DNA polyploidy. To functionally evaluate induced megakaryocytes, these cells were transplanted into sublethally irradiated NOD/SCID mice. Viability and cell being of these mice were monitored after injection. Mice of the negative control group (n=3) were injected with saline. In the experimental group (n=6), each mouse was injected with 1.0×10⁷ cells from the second stage of culture.

Results: After the first stage culture, proliferation folds of total cells and CD34⁺ cells were 85.65±7.03 and 62.91±4.36, respectively. The calculated yield from each CD34⁺ cell was between 1.0×10⁴ to 1.5×10⁴ CD41⁺ megakaryocytes with a purity of CD41⁺ and CD42⁺ cells reaching 93.7%±2.8% and 80.3%±5.8%, respectively. Differentiated cells were morphologically discernible as they were much larger than starting CD34⁺ cells with apparent lobular nuclei and numerous α-granules. In addition, about 32.67%±7.43% of induced megakaryocytes exhibited 4N or larger DNA content (4N 17.6%±4.12%; 8N 10.93%±2.48%; >8N 3.23%±1.34%). In mouse studies, samples collected from the negative control group contained no cells positive for human CD41a and CD42b markers. In the experimental group, human platelets were detected in mouse peripheral blood 3 days post-transplantation. At day 14 post-transplantation, the percentage of platelets derived from injected human megakaryocytes reached 13.6%±6.2%. Human megakaryocytes were also detected in mouse bone marrow 7 days post-transplantation, peaking at day 14 (~2.38% of total bone marrow megakaryocytes).

Conclusions: We have established a stem cell expansion and differentiation platform that can be adapted to large-scale production of mature megakaryocytes from umbilical cord blood cells. Significantly, induced megakaryocytes are capable of engrafting in mouse bone marrow and producing platelets after transplantation into irradiated NOD/SCID mice. Therefore, our experimental platform is capable of producing a sufficient number of functional megakaryocytes for various clinical applications in the future.