Hemogenic Endothelium-like Changes Were Derived to an Adult Human Dermal Fibroblast By Electrical Stimulation

Background and Aims: We previously reported that when an adult human dermal fibroblast (HDF) was cultured with interleukin (IL)-1-beta (b) and erythropoietin (EPO), hematopoiesis-related molecules expressed. And, lymphatic duct-neogenesis genes also expressed, and vascular endothelial growth factor (VEGF)-A, and -C were produced. When anti-human VEGF-C antibody (Ab) was added to the cultures, hematopoiesis-related genes expressed; however, morphologic changes were not observed (54^th ASH, 18^th EHA). Reported findings on nuclear transfer reveal that electrical stimulation induces nuclear fusion and changes cell-fate. We observed the effect of electrical stimulation to IL-1-b-stimulated HDF.

Materials and Methods: HDF was cultured with IL-1-b, EPO, VEGF-A, and anti-human VEGF-C Ab for 14 days. Then, cells were suspended in electrical stimulation-buffer (0.25 M d-sorbitol, 0.1 mM Ca-acetate, 0.5 mM Mg-acetate, 1 mg/mL fatty-acid-free BSA, 0.5 mM HEPES), and incubated on ice for 10 minutes. Cells were stimulated at 110V 20mA (0.2 cm electrode, gap), and were further cultured with k/o DMEM containing 20% KSR and SCF, IL-6, FL, IGF-2, and VEGF-A. Morphological changes and expressions of vascular endothelial cell-related genes, and hematopoiesis-related ones were observed.

Results: When HDF was stimulated electrically, a few cells showed vascular endothelial cell-morphology after two days. When cells were further cultured in a hematopoietic stem cell-culturing condition, a part of endothelial cell-morphology changed to that of non-adherent hematopoietic cells or blastic colonies, in which increased expression-levels of SCL, CD41, GATA-2, CD34, and CD45 were accepted with significant statistical difference.

Discussion: Recent reports reveal that a kind of vascular endothelial cells, called hemogenic endothelium, can convert to a hematopoietic cell, and works an important role in hematopoietic stem cell-generation. We hypothesized that when cells expressed a kind of specific transcripts, some stimulation triggerred to convert morphologic changes and cell-fate as are observed in nuclear fusion. And, our observations indicated that when IL-1-b-stimulated HDF, expressing hematopoiesis-related transcripts, were stimulated electrically, cellular morphology changed. Currently, we are precisely analyzing electrically stimulated HDF’s biological characteristics.