Chapter 27
Isolation, Cryopreservation, and Characterization
of iPSC-Derived Megakaryocytes
Denys Pogozhykh, Rainer Blasczyk, and Constanc ¸ a Figueiredo
Abstract
Current research in the field of transfusion medicine is focused on developing innovative approaches to
generate populations of functional megakaryocytes (MKs) ex vivo. This may open perspectives to establish
alternative therapies for donor platelet transfusion in the management of thrombocytopenic patients and
pave the way for novel regenerative approaches. Efficient cryopreservation techniques can provide the
opportunity for long-term storage and accumulation of necessary amounts of MKs in a ready-to-use
manner. However, in this case, besides the viability, it is crucial to consider the recovery of functional
MK properties after the impact of freezing. In this chapter, the possibility to cryopreserve iPSC-derived
MKs is described. In particular, the methods for a comprehensive analysis of phenotypic and functional
features of MKs after cryopreservation are proposed. The use of cryopreserved in vitro-produced MKs may
benefit to the field of transfusion medicine to overcome the lack of sufficient blood donors.
Key words Megakaryocytes, Platelets, Cryopreservation, Proplatelets, Polyploidy
1 Introduction
Transfusion of donor platelets (PLTs) is often complicated by
significantly limited availability, immunological concerns, expensive
and sophisticated donation procedures, contamination and quality
issues, as well as the short shelf life of donated material [1–3]. To
overcome these issues, current research is directed on possibilities
to generate populations of functional MKs and PLTs from various
sources ex vivo [1–9]. Induced pluripotent stem cell (iPSC) blood
farming technologies allow generation of functional iPSC-derived
MKs, also in clinically relevant large-scale quantities [10–14]. This
opens perspectives for the application of iPSC-derived MKs as an
additional or alternative approach to transfusion of donated PLTs.
At the same time, future clinical implementation of iPSC-derived
MK/PLT transfusion will require methods to ensure availability of
supplies in a ready-to-use manner in sufficient amounts and functional form. Advancing technologies of efficient cryopreservation
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_27, © Springer Science+Business Media, LLC, part of Springer Nature 2021
539
Isolation, Cryopreservation, and Characterization
of iPSC-Derived Megakaryocytes
Denys Pogozhykh, Rainer Blasczyk, and Constanc ¸ a Figueiredo
Abstract
Current research in the field of transfusion medicine is focused on developing innovative approaches to
generate populations of functional megakaryocytes (MKs) ex vivo. This may open perspectives to establish
alternative therapies for donor platelet transfusion in the management of thrombocytopenic patients and
pave the way for novel regenerative approaches. Efficient cryopreservation techniques can provide the
opportunity for long-term storage and accumulation of necessary amounts of MKs in a ready-to-use
manner. However, in this case, besides the viability, it is crucial to consider the recovery of functional
MK properties after the impact of freezing. In this chapter, the possibility to cryopreserve iPSC-derived
MKs is described. In particular, the methods for a comprehensive analysis of phenotypic and functional
features of MKs after cryopreservation are proposed. The use of cryopreserved in vitro-produced MKs may
benefit to the field of transfusion medicine to overcome the lack of sufficient blood donors.
Key words Megakaryocytes, Platelets, Cryopreservation, Proplatelets, Polyploidy
1 Introduction
Transfusion of donor platelets (PLTs) is often complicated by
significantly limited availability, immunological concerns, expensive
and sophisticated donation procedures, contamination and quality
issues, as well as the short shelf life of donated material [1–3]. To
overcome these issues, current research is directed on possibilities
to generate populations of functional MKs and PLTs from various
sources ex vivo [1–9]. Induced pluripotent stem cell (iPSC) blood
farming technologies allow generation of functional iPSC-derived
MKs, also in clinically relevant large-scale quantities [10–14]. This
opens perspectives for the application of iPSC-derived MKs as an
additional or alternative approach to transfusion of donated PLTs.
At the same time, future clinical implementation of iPSC-derived
MK/PLT transfusion will require methods to ensure availability of
supplies in a ready-to-use manner in sufficient amounts and functional form. Advancing technologies of efficient cryopreservation
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_27, © Springer Science+Business Media, LLC, part of Springer Nature 2021
539