Large-scale production of megakaryocytes in microcarrier-supported stirred suspension bioreactors.

Megakaryocytes (MKs) are the precursors of platelets (PLTs) and may be used for PLT production in vivo or in vitro, as well as a source for PLT-derived growth factors. Induced pluripotent stem cells represent an unlimited cell source for the in vitro production of MKs. This study aimed at developing an effective, xeno-free and scalable system to produce high numbers of MKs. In particular, microcarrier beads-assisted stirred bioreactors were evaluated as a means of improving MK yields. This method resulted in the production of 18.7 × 107 MKs per 50 ml medium. Laminin-coated microcarriers increased MK production per iPSC by up to 10-fold. MKs obtained in this system showed typical features of mature MKs and were able to produce PLTs in vitro and in vivo. To increase safety, MKs produced in the bioreactors were irradiated; a procedure that did not affect their capability to form proPLTs and PTLs after transfusion. In vitro generated MKs represent a promising alternative to donor PLTs and open the possibility for the development of innovative MK-based cell therapies.

Characterization of induced pluripotent stem cell-derived megakaryocyte lysates for potential regenerative applications.

Recently, platelet-derived growth factors present in lysates became an extreme interest in the field of regenerative medicine such as in wound healing and as substitutes to foetal bovine serum in xeno-free cell culture systems. However, the generation of such platelet lysates completely depends on the availability of platelet donors. In this study, the possibility to use in vitro-generated megakaryocytes derived from induced pluripotent stem cells (iPSCs) as a cell source for typical platelet growth factors was investigated. Therefore, the presence and levels of those factors were characterized in in vitro-produced megakaryocytes. In comparison with platelets, in vitro-generated megakaryocytes showed a multifold increased content in transcript and protein levels of typical platelet growth factors including platelet-derived growth factors (PDGFs), transforming growth factor (TGF)-1ß, vascular endothelial cell factor (VEGF)-A, epidermal growth factor (EGF), insulin-like growth factor (IGF)-1 and tissue factor (TF). Hence, iPSC-derived megakaryocytes may serve as an efficient cell source for a donor-independent generation of growth factor-rich lysates with a broad application potential in innovative cell culture systems and regenerative therapies.

Towards the Manufacture of Megakaryocytes and Platelets for Clinical Application.

Platelet transfusions are used in standard clinical practice to prevent hemorrhage in patients suffering from thrombocytopenia or platelet dysfunctions. Recently, a constant rise on the demand of platelets for transfusion has been registered. This may be associated with several factors including demographic changes, population aging as well as incidence and prevalence of hematological diseases. In addition, platelet-regenerative properties have been started to be exploited in different areas such as tissue remodeling and anti-cancer therapies. These new applications are also expected to increase the future demand on platelets. Thus, in vitro generated platelets may constitute a highly desirable alternative to meet the rising demand on platelets. Several factors have been considered in the road trip of producing in vitro megakaryocytes and platelets for clinical application. From selection of the cell source, differentiation protocols and culture conditions to the design of optimal bioreactors, several strategies have been proposed to maximize production yields while preserving functionality. This review summarizes new advances in megakaryocyte and platelet differentiation and their production upscaling.

Autocrine GM-CSF transcription in the leukemic progenitor cell line KG1a is mediated by the transcription factor ETS1 and is negatively regulated through SECTM1 mediated ligation of CD7.

BACKGROUND: CD7 expression is found on ~30% of acute myeloblastic leukemias (AML). The leukemic progenitor cell line KG1a (CD7+) constitutively expresses GM-CSF while the parental KG1 (CD7-) cell line does not. This study focuses on the molecular basis of CD7 mediated GM-CSF regulation. METHODS: KG1a cells were treated with recombinant SECTM1-Fc protein, the PI3K kinase inhibitors wortmannin, LY292004, or PI4K activator spermine. Stable KG1-CD7+, KG1a-shCD7, KG1a-shETS1 as well as KG1a-GFP, KG1a-PKCßII-GFP cell lines were generated and the levels of CD7, GM-CSF and ETS-1 mRNA and protein were compared by real-time-PCR, western blotting, flow cytometry and ELISA. RESULTS: SECTM1 is expressed in Human Bone Marrow Endothelial Cells (HBMEC) and its expression can be upregulated by both IFN-γ. KG1a cells demonstrated high expression levels of CD7 and ETS-1 allowing a constitutative signaling through the PI3K/Atk pathway to promote GM-CSF expression, while KG1 cells with low expression of CD7 and ETS-1 showed low GM-CSF expression. On KG1a cells GM-CSF expression could be negatively regulated by PI3K inhibitors or by recombinant SECTM1-Fc. Overexpression of CD7 in KG1 cells was insufficient to promote GM-CSF expression, while silencing of CD7 or ETS-1 resulted in reduced GM-CSF expression levels. Differentiation capable KG1a cells overexpressing PKCßII illustrated complete loss of CD7, but maintained normal levels of both ETS-1 and GM-CSF expression. CONCLUSION: These findings add an additional layer to the previously described autocrine/paracrine signaling between leukemic progenitor cells and the bone marrow microenvironment and highlight a role for SECTM1 in both normal and malignant hematopoiesis. GENERAL SIGNIFICANCE: This work shows that SECTM1 secreted from bone marrow stromal cells may interact with CD7 to influence GM-CSF expression in leukemic cells.