ABSTRACT
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.
Subject(s)
Antigens, CD34/genetics , Automation, Laboratory/methods , Flow Cytometry/methods , Myocardial Infarction/therapy , Peripheral Blood Stem Cell Transplantation/methods , Peripheral Blood Stem Cells/cytology , Adult , Animals , Antigens, CD34/metabolism , Cells, Cultured , Clinical Trials as Topic , Humans , Immunophenotyping/methods , Male , Middle Aged , Peripheral Blood Stem Cells/metabolism , Primary Cell Culture/methods , RatsABSTRACT
Improvements of microarray techniques for genotyping purposes have focused on increasing the reliability of this method. Here we report the development of a genotyping method where a microarray was spotted with stemloop probes, especially designed to optimize the hybridization specificity of complementary DNA sequences. This accurate method was used to screen for four common disease-causing mutations involved in a neurological disorder called Charcot-Marie-Tooth disease (CMT). Healthy individuals' and patients' DNA were amplified and labeled by PCR and hybridized on microarray. The spot signal intensities were 81 to 408 times greater for perfect compared with mismatched target sequences, differing by only one nucleotide (discrimination ratio) for healthy individual "homozygous" DNA. On the other hand, "heterozygous" mutant DNA samples gave rise to signal intensity ratios close to 1, as expected. The genotypes obtained by this method were perfectly consistent with those determined by direct PCR sequencing. Cross-hybridization rates were very low, resulting in further multiplexing improvements. In this study, we also demonstrated the feasibility of real-time hybridization detection of labeled synthetic oligonucleotides with concentrations as low as 2.5 nM.
Subject(s)
DNA Mutational Analysis/methods , Oligonucleotide Array Sequence Analysis/methods , Point Mutation/genetics , Base Sequence , Case-Control Studies , Charcot-Marie-Tooth Disease/genetics , Humans , Molecular Sequence Data , Nucleic Acid Conformation , Oligonucleotides/chemistry , Oligonucleotides/genetics , Sensitivity and SpecificityABSTRACT
We previously developed a highly specific method for detecting SNPs with a microarray-based system using stem-loop probes. In this paper we demonstrate that coupling a multiplexing procedure with our microarray method is possible for the simultaneous detection and genotyping of four point mutations, in three different genes, involved in Charcot-Marie-Tooth disease. DNA from healthy individuals and patients was amplified, labeled with Cy3 by multiplex PCR; and hybridized to microarrays. Spot signal intensities were 18 to 74 times greater for perfect matches than for mismatched target sequences differing by a single nucleotide (discrimination ratio) for "homozygous" DNA from healthy individuals. "Heterozygous" mutant DNA samples gave signal intensity ratios close to 1 at the positions of the mutations as expected. Genotyping by this method was therefore reliable. This system now combines the principle of highly specific genotyping based on stem-loop structure probes with the advantages of multiplex analysis.