Use of IL3 and chromatin-modifying reagents valproic acid and 5-aza-2′-deoxycytidine to affect mobilized peripheral blood CD34+cell fate decisions

摘要

Background and objectives In 2006 Shinya Yamanaka successfully reprogrammedudmouse fibroblasts back to an embryonic stem cell-like state (called inducedudpluripotent cells, iPS cells) using retrovirus to introduce four genes thatudencode critical transcription factor proteins (Oct4, Sox2, KLF4, and c-Myc). Thisudability to reprogram has promising future applications in clinical and biomedicaludresearch for study of diseases, development of candidate drugs and to supportudtherapeutic treatments in regenerative medicine. However, the clinical applicationsudhave to meet GMP requirements without the risk of insertional mutagenesisudassociated with retrovirus. Chromatin modifying agents are widely used in manyudprotocols to generate iPS cells and culture of blood CD34+ cells with chromatin-modifyingudagents can lead to an increase in marrow repopulating cells and in theudcase of valproic acid increased erythroid cell colony formation. We undertookudresearch to help understand what effects these reagents have on mobilised peripheraludblood (mPB) CD34+ cells and optimised the expansion medium protocoludto facilitate reprogramming work. This project aims to utilize peripheral bloodudmononuclear cells (MNC), one of the most easily accessible tissues to generate iPSudcells using an efficient non-viral, feeder cell free methodology, with the ultimateudgoal of moving this methodology towards clinical use.udMaterials and Methods G-CSF mobilised peripheral blood, buffy coat, cordudblood and fetal liver were obtained from patients and donors under informedudconsent and ethics committee approval. Haematopoietic stem/progenitor cellsudCD34+ or CD133+) isolated by magnetic separation were flow cytometry sortedudinto CD34+/CD133+, CD34+/CD133-, and CD34-/CD133+ sub-populations andudtheir lineage potential were assessed in colony forming unit assays. The effectudof epigenetic modifiers valproic acid and 5-aza-2-deoxycytidine used singly or inudcombination with each other and with IL3 on phenotype and lineage potentialudof cultured CD34+ cells from mobilised peripheral blood were assessed by flowudcytometry and colony-forming unit assays. Prior to reprogramming mononuclear cells from peripheral blood or CD34+udcells from blood were expanded in culture medium supplemented with stemudcell factor (SCF), Fms-related tyrosine kinase 3 ligand (Flt3L) and Interleukin-ud3 (IL-3) for several days. Actively proliferating cells were reprogrammed byudelectroporation using episomal vectors with an oriP/EBNA-1 backbone to deliverudfive reprogramming genes, Oct4, Sox2, Lin28, L-Myc, and Klf4. Electroporatedudcells were seeded onto matrigel coated plates immediately after transfection orudwere reseeded after three days’ culture. Subsequently, cells were cultured inudspecific medium on different days. When iPS colonies appeared, they wereudpicked and cultured as for ES cells. Once established, iPS cell lines wereudimmunophenotyped using flow cytometry and immunofluorescence and theirudpotential to differentiate into the three germ layers was assessed in vitro.udResults and Conclusion The largest subpopulation of CD34+ cells wasudCD34+/CD133+ population which was essentially committed to myeloid colonyudproduction, while much smaller CD34+/CD133- subpopulation had a greater capacityudto generate erythroid colonies. Optimised cytokine cocktail for expansionudof CD34+ cells included IL-3, important in improving expansion and maintainingudfunctionality of CD34+ cells. The optimised cytokine cocktail comprised 100udng/ml SCF, 10 ng/ml Flt3L, and 20 ng/ml IL-3, which maintained CD34+ cellsudand MNC in an active proliferating state. In addition, valproic acid and IL3 wereudfound to act synergistically, to increase the numbers of CD34+/CD36+ positiveudcells. However, we found that an apparent increase in red cell colony formationudactually resulted from a decrease in white cell colonies, so no overall increase inudred cell colonies was seen when equivalent numbers of CD34+ cells were plated.udProliferating MNC maintained in optimised cytokine cocktail were amenableudto electroporation for the effective delivery of episomal transcription factorsud(Oct4, Sox2, Klf4, L-Myc, and Lin28) within a backbone of oriP/EBNA-1. Weudsuccessfully developed an efficient and simple method for reprogramming MNCudfrom fresh or frozen samples to generate induced pluripotent cells using episomaludvectors in a feeder-free system without any requirement for small molecules andudthe highest reprogramming efficiency is 0.033% (65 colonies from 2 ◊ 105 seedingudMNC). The cytokine cocktail and reprogramming methods work better in CD34+udcells from cord blood or fetal liver, and we obtained 148 iPS colonies from 105udseeding cells (0.148%) at most. In addition, fibroblasts from adult and fetal liverudcan be successfully reprogrammed using the same reprogramming method. Theuduse of episomal vectors with an oriP/EBNA-1 backbone to deliver reprogrammingudgenes, and efficient electroporation were the most important factors in efficiencyudof the reprogramming process. In addition, it is pivotal to initiate transfectionudwhen cells are actively proliferating. The iPS cell lines we generated maintained the successful expression of ES markers including Oct4, Nanog, SSEA3. SSEA4,udTRA-1-60 and TRA-1-81, and had the capacity to successfully differentiate intoudcell types of ectoderm, mesoderm and endoderm layers in vitro.