Development of a Clinical Grade Procedure for Generation of mRNA Transfected Dendritic Cells from Purified Frozen CD34+ Blood Progenitor Cells

Abstract

Enriched CD34+ peripheral blood progenitor cells (PBPC) are frequently used as stem cell support in cancer patients following high dose therapy. Since precursor dendritic cells (DCs) originate from haematopoietic progenitor cells, purified CD34+ cells might also serve as starting cells for ex-vivo production of DC. In the present study we developed a clinical grade procedure for ex-vivo production of DC derived from enriched CD34+ cells. Different concentrations of CD34+ cells were grown in gas-permeable Teflon bags with different serum-free and serum-containing media supplemented with GM-CSF, IL-4, TNF-ex, SCF, Flt-3L and INF-ex. Serum-free CellGroSCGM medium for 7 days followed by CellGroDC medium in 7 days gave the same results as serum-containing medium. After incubation the cultured cells containing immature DCs were concentrated and transfected with tumour mRNA from human prostate cancer cell lines employing a highly efficient electroporation procedure. Thawed transfected DCs were able to elicit primary T-cell responses in vitro against antigens encoded by the prostate cancer mRNA as shown by ELlSPOT assay using mocktransfected DCs as control. Our results show that frozen enriched CD34+ cells can be an alternative and efficient source for production of DCs for therapeutic purpose.Dendritic cells (DCs) are the most powerful antigen-presenting cells (APe) specialized to initiate and regulate immune responses (1-2). The clinical use as cellular adjuvants in vaccination strategies has been aided by the development of methodologies to generate these cells on a large scale in culture. DCs can be grown ex-vivo from blood monocytes (3-5) or enriched CD34+ progenitors (6-7), using combinations of several cytokines/growth factors. Since we routinely use in our laboratory enriched CD34+ stem cells as stem cell support following high dose radio-and chemotherapy it was of interest to test if such cells could also be applied for vaccine purposes (8)(9), with a long term strategy of combining the two forms of therapy.Some published data indicate that CD34+ derived DC may work more efficiently as APC than those derived from monocytes (10), and recent data indicate that vaccine programs using CD34+ cell derived DCs results in improved clinical results (11). But most in vitro culture systems for production of DCs include serum (9-12). Since DCs are able to take up and process serum-derived antigens that are present in the cell cultures, such DC can, when injected create unwanted reactions in the patients, in particular when fetal calf serum (PCS) is used. Serum-free culturing condition is thus preferable, but in most previous culture experiments these conditions resulted in a lower yield of DCs. (13)(14).

Dental Pulp Stem Cells Bioadhesivity: Evaluation on Mineral-Trioxide-Aggregate

“…Moreover, dental pulp is a viable source of easily attainable cells with a possible potential for cell transplantation therapies, indicating the presence of a cell population in the dental pulp with neuronal differentiation capacity (21), and also having the ability to differentiate in mesenchymal tissue, after induction, offering a novel approach for regenerative medicine (22)(23)(24). These results are in accordance with the existence in the adult organism of a biological niche of the mesenchymal stem cells that are not only restricted to bone marrow but are present in different tissues and, under defined inductive conditions, are able to acquire characteristics of cells derived from embryonic mesoderm, as well as possessing ectodermal and neuronal properties (25)(26)(27). In this paper, apart from cytofluorimetric determination, we report the isolation and characterization, by morphological analysis, of a homogeneous mesenchymal stem cells population expanded ex vivo from dental pulp and their ability to grow in the presence of biomaterials.…”

Cryopreservation of adenovirus-transfected dendritic cells (DCs) for clinical use

“…Although satisfactory and clinically applicable cryopreservation methods for DCs have been described [18][19][20][21], methods to cryopreserve products transduced with Adv vectors have been little studied [22]. In most clinical trials reported to date, DCs were freshly prepared for each administration either from fresh blood [23][24][25][26][27], leukapheresis products [23,28,29], or from frozen aliquots of PBMCs obtained by leukapheresis [27].…”

Dendritic Cells as Pharmacological Tools for Cancer Immunotherapy

“…Cell types frequently used as starting material for DC vaccine manufacturing are monocytes (Anguille et al, 2012c) and CD34 + hematopoietic progenitor cells derived from bone marrow (Lardon et al, 1997), peripheral blood (Mu et al, 2004), or cord blood (Chen et al, 2013). Monocyte-derived DCs (moDCs) are by far the most commonly used DC type in DC vaccine formulations Chiang et al, 2015).…”