Efficient Induction of CD34+ Progenitor Cells From Human Bone Marrow Mesenchymal Stem Cell-Derived Induced Pluripotent Stem Cells with Defined Chemicals

Abstract 2347

Induced pluripotent stem cells (iPSCs) hold great potential in clinical application with indefinite passages, pluripotency of forming three germ layers and immunological compatibility with patients. Human bone marrow mesenchymal stem cell-derived iPSCs (hBMMSC-iPSCs) may have more virtues than other types of cell–derived iPSCs do in diseases treatment for their wide application in clinic for many years. Efficient differentiation of induced pluripotent stem cells (iPSCs) to a specialized cell type is a crucial step for iPSC clinical application. CD34+ progenitor cells derived from hBMMSC-iPSCs by treatment with defined chemicals have not been reported. Here, base on the efficient generation of hBMMSC-iPSCs with cDNA encoding four transcription factors OCT4, SOX2, KLF4 and c-MYC, and the compounds of p53 siRNA, VPA and Vc, we proposed that CD34+ progenitor cells, retaining hematopoietic and endothelial cell potential, could be efficiently obtained from hBMMSC-iPSCs by treatments with chemicals exerting function on mesoderm and hematopoietic cells. The differentiated hBMMSC-iPSCs treated with the chemicals comprised nearly 20% proportion of CD34+ progenitor cells, about 20-fold increasing compared to the spontaneous differentiation hBMMSC-iPSCs and higher than that from human skin fibroblast-derived iPSCs (hFib-iPSCs) (nearly 15%) and human embryonic stem cell line H1 (nearly 17%) by flow cytometry analysis. Those cells retained the hematopoietic and endothelial cell potential as CD34+ progenitor cells derived from umbilical cord blood. After coculture with the first chemical cocktails including BMP4 and SCF for 5 days, a higher proportion of the mesoderm cells were induced from hBMMSC-iPSCs than that from the untreated groups (P<0.05) by analysis the expression of Brachyury and GATA2 with RT-PCR and immunochemistry analysis. The efficient mesoderm cell activity was then directed to CD34+ progenitor cells with the following chemicals as SCF and IL-3 for another 7–9 days by analysis of cellular phenotype and the expression of the hematopoietic transcription factors TAL-1 and GATA-2. Sorted by CD34 magnetic separation and exposed to semisolid media with the hematopoietic cytokines including EPO and G-CSF, CD34+ progenitor cells formed various hematopoietic colonies as BFU-E, CFU-E, CFU-G, CFU-GM, and CFU-GEMM. CFUs derived from hBMMSC-iPSC groups treated with the chemicals outnumbered those from hFib-iPSCs, the spontaneous differentiation hBMMSC-iPSCs and H1 groups. Hematopoietic cell lineages as erythroid, granulocyte, and megakaryocyte were identified with Wright-Giemsa staining. During the hematopoietic commitment of hBMMSC-iPSCs, the pluripotent gene Oct4 continued to be down-regulated and completely vanished at day 14 after the induction. However, the expression of the hematologic transcription factor GATA-2 experienced a dynamic process with up-regulation at the initial induction stage and then gradual down-regulation after CD34+ progenitor cell commitment to CFU forming. Endothelial cell potential of CD34+ progenitor cells was also verified by demonstration of the vascular tube-like structure formation and the expression of endothelial specific markers CD31 and VE-CADHERIN. The efficient induction of CD34+ progenitor cells from hBMMSC-iPSCs with defined chemicals, avoiding contamination from indentified factors with using mouse feeder lays or other poorly defined induction components, gives an opportunity for the generation of unlimited HLA matched transplantable cells, provides cell models for study the mechanism of haemopoiesis and related diseases, and therefore promotes iPSC clinical application.