Design and Validation of an Automated Process for the Expansion of Peripheral Blood-Derived CD34+ Cells for Clinical Use After Myocardial Infarction.

We previously demonstrated that intracardiac delivery of autologous peripheral blood-derived CD34+ stem cells (SCs), mobilized by granulocyte-colony stimulating factor (G-CSF) and collected by leukapheresis after myocardial infarction, structurally and functionally repaired the damaged myocardial area. When used for cardiac indication, CD34+ cells are now considered as Advanced Therapy Medicinal Products (ATMPs). We have industrialized their production by developing an automated device for ex vivo CD34+ -SC expansion, starting from a whole blood (WB) sample. Blood samples were collected from healthy donors after G-CSF mobilization. Manufacturing procedures included: (a) isolation of total nuclear cells, (b) CD34+ immunoselection, (c) expansion and cell culture recovery in the device, and (d) expanded CD34+ cell immunoselection and formulation. The assessment of CD34+ cell counts, viability, and immunophenotype and sterility tests were performed as quality tests. We established graft acceptance criteria and performed validation processes in three cell therapy centers. 59.4 × 106 ± 36.8 × 106 viable CD34+ cells were reproducibly generated as the final product from 220 ml WB containing 17.1 × 106 ± 8.1 × 106 viable CD34+ cells. CD34+ identity, genetic stability, and telomere length were consistent with those of basal CD34+ cells. Gram staining and mycoplasma and endotoxin analyses were negative in all cases. We confirmed the therapeutic efficacy of both CD34+ -cell categories in experimental acute myocardial infarct (AMI) in immunodeficient rats during preclinical studies. This reproducible, automated, and standardized expansion process produces high numbers of CD34+ cells corresponding to the approved ATMP and paves the way for a phase I/IIb study in AMI, which is currently recruiting patients. Stem Cells Translational Medicine 2019;8:822&832.

ONSL and OSKM cocktails act synergistically in reprogramming human somatic cells into induced pluripotent stem cells.

The advent of human induced pluripotent stem cells (hiPSC) is revolutionizing many research fields including cell-replacement therapy, drug screening, physiopathology of specific diseases and more basic research such as embryonic development or diseases modeling. Despite the large number of reports on reprogramming methods, techniques in use remain globally inefficient. We present here a new optimized approach to improve this efficiency. After having tested different monocistronic vectors with poor results, we adopted a polycistronic cassette encoding Thomson's cocktail OCT4, NANOG, SOX2 and LIN28 (ONSL) separated by 2A peptides. This cassette was tested in various vector backbones, based on lentivirus or retrovirus under a LTR or EF1 alpha promoter. This allowed us to show that ONSL-carrier retrovectors reprogrammed adult fibroblast cells with a much higher efficiency (up to 0.6%) than any other tested. We then compared the reprogramming efficiencies of two different polycistronic genes, ONSL and OCT4, SOX2, KLF4 and cMYC (OSKM) placed in the same retrovector backbone. Interestingly, in this context ONSL gene reprograms more efficiently than OSKM but OSKM reprograms faster suggesting that the two cocktails may reprogram through distinct pathways. By equally mixing RV-LTR-ONSL and RV-LTR-OSKM, we indeed observed a remarkable synergy, yielding a reprogramming efficiency of >2%. We present here a drastic improvement of the reprogramming efficiency, which opens doors to the development of automated and high throughput strategies of hiPSC production. Furthermore, non-integrative reprogramming protocols (i.e. mRNA) may take advantage of this synergy to boost their efficiency.