In vitro biological characteristics of human cord blood-derived megakaryocytes.
BACKGROUND
Umbilical cord blood (CB) has been used as an alternative source for haematopoietic stem cell transplantation (HSCT) in recent years. However, delayed platelet recovery is frequently associated with CB HSCT. Megakaryocytes (Mk) are the specialised precursors of platelets and they are among the rarest haemopoietic cell types. Despite the rapid expansion of our knowledge of megakaryopoiesis in recent years, many questions, such as the molecular regulatory mechanisms in Mk differentiation and maturation, platelet formation and release, remain unanswered in CB-derived megakaryopoiesis. Variations can be seen from the literature by individual investigators using different approaches for Mk-specific differentiation and maturation induction. The development of in vitro culture methods to obtain sufficient numbers of Mks from readily available haematopoietic stem cells is of value for both basic research and clinical applications.


METHODS
The CD34+ cells from cord blood samples were cultured in serum-free medium with haematopoietic growth factors (GFs), such as IL-3, stem cell factor (SCF), and thrombopoietin (Tpo). The differentiation of Mk was monitored using Mk- and platelet-specific monoclonal antibodies and flow cytometric analysis. The morphology of the cultured cells was studied by both light and electronic microscopy (LM and EM). The involvement of the human Notch gene family members was studied by real time-polymerase chain reaction (RT-PCR). Maturation of the cultured Mks was studied using flow cytometric analysis for both platelet-specific surface markers and enodomitosis. Platelet activation was assessed in the cytoplasmic fragments harvested from the cultures.


RESULTS
Specific Mk differentiation of >70% resulted from a 2-step culture approach using IL-3, SCF and Tpo for 7 days followed by Tpo only for another 14 days. RT-PCR showed high-level expression of both Notch-1 and its ligand, Jagged-1, in the cultured Mks. Limited levels of polyploidy (>4N, endomitosis, EnM) were observed in the cultured Mks. The results also showed that the cytoplasmic fragments from the cultures responded to platelet activation reagents, including ADP and collagen, marked by upregulation of platelet-specific activation markers, such as CD62P (P-selectin) and PAC-1 (gpalphaIIbbeta3).


CONCLUSIONS
The methods used in this study are specific for differentiation of Mk from CB CD34+ cell, which can partially mature and produce functional platelets in vitro. This approach for human Mk differentiation could be further optimised and may be adapted on larger scales for clinical purposes.
Sun
L
L
Department of Clinical Research, Singapore General Hospital, Singapore. gcrsl@sgh.com.sg
Tan
P
P
Yap
C
C
Hwang
W
W
Koh
L P
LP
Lim
C K
CK
Aw
S E
SE
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D018952:Antigens, CD34 / Q000097:blood*; D001483:Base Sequence; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D048429:Cell Size; D003470:Culture Media; D005312:Fetal Blood / Q000166:cytology*; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology*; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D018952:Antigens, CD34; D003470:Culture Media