Establishment of Mouse Embryonic Stem Cell-Derived Erythroid Progenitor Cell Lines Able to Produce Functional Red Blood Cells

BACKGROUND: The supply of transfusable red blood cells (RBCs) is not sufficient in many countries. If erythroid cell lines able to produce transfusable RBCs in vitro were established, they would be valuable resources. However, such cell lines have not been established. To evaluate the feasibility of establishing useful erythroid cell lines, we attempted to establish such cell lines from mouse embryonic stem (ES) cells. METHODOLOGY/PRINCIPAL FINDINGS: We developed a robust method to obtain differentiated cell lines following the induction of hematopoietic differentiation of mouse ES cells and established five independent hematopoietic cell lines using the method. Three of these lines exhibited characteristics of erythroid cells. Although their precise characteristics varied, each of these lines could differentiate in vitro into more mature erythroid cells, including enucleated RBCs. Following transplantation of these erythroid cells into mice suffering from acute anemia, the cells proliferated transiently, subsequently differentiated into functional RBCs, and significantly ameliorated the acute anemia. In addition, we did not observe formation of any tumors following transplantation of these cells. CONCLUSION/SIGNIFICANCE: To the best of our knowledge, this is the first report to show the feasibility of establishing erythroid cell lines able to produce mature RBCs. Considering the number of human ES cell lines that have been established so far, the intensive testing of a number of these lines for erythroid potential may allow the establishment of human erythroid cell lines similar to the mouse erythroid cell lines described here. In addition, our results strongly suggest the possibility of establishing useful cell lines committed to specific lineages other than hematopoietic progenitors from human ES cells

Erythroid-Specific Expression of β-globin from Sleeping Beauty-Transduced Human Hematopoietic Progenitor Cells

Gene therapy for sickle cell disease will require efficient delivery of a tightly regulated and stably expressed gene product to provide an effective therapy. In this study we utilized the non-viral Sleeping Beauty (SB) transposon system using the SB100X hyperactive transposase to transduce human cord blood CD34+ cells with DsRed and a hybrid IHK–β-globin transgene. IHK transduced cells were successfully differentiated into multiple lineages which all showed transgene integration. The mature erythroid cells had an increased β-globin to γ-globin ratio from 0.66±0.08 to 1.05±0.12 (p = 0.05), indicating expression of β-globin from the integrated SB transgene. IHK–β-globin mRNA was found in non-erythroid cell types, similar to native β-globin mRNA that was also expressed at low levels. Additional studies in the hematopoietic K562 cell line confirmed the ability of cHS4 insulator elements to protect DsRed and IHK–β-globin transgenes from silencing in long-term culture studies. Insulated transgenes had statistically significant improvement in the maintenance of long term expression, while preserving transgene regulation. These results support the use of Sleeping Beauty vectors in carrying an insulated IHK–β-globin transgene for gene therapy of sickle cell disease

Genetic programming of macrophages generates an in vitro model for the human erythroid island niche

In vitro differentiation of red blood cells (RBCs) is a desirable therapy for various disorders. Here the authors develop a culture system using stem cell-derived macrophages to show that inducible expression of a transcription factor, KLF1, enhances RBC production, potentially through the induction of three soluble factors, ANGPTL7, IL33 and SERPINB2

Transcollation technique in the thoracoscopic treatment of primary spontaneous pneumothorax

OBJECTIVES: The already low invasiveness of the thoracoscopic treatment of spontaneous pneumothorax may be further reduced by the transcollation® technique. Herein, we report our further experience with a new device, to coagulate blebs and bullae, compared with contrastto endostapler resection. METHODS: Data of patients with recurrent or persistent spontaneous pneumothorax, who underwent thoracoscopic treatment, were prospectively collected and reviewed. Those with blebs or bullae (Stages III and IV in accordance with Vanderschueren’s classification) were treatedwithanewdevice,basedoncouplingsalinesolutionperfusionwithradiofrequencyenergy.Thecombinationof fluidwithradiofrequencyallowsthesealing of tissue, avoiding charring or burning.Mostoperationswere performed through two1-cmincisions only. RESULTS: From 2005 to 2010, 73 patients were treated. These were 59 males (80.8%) and 14 females (19.2%), with a mean age of 27.9 years[standarddeviation(SD):11.7].Forty-threepatientsunderwentgeneral anaesthesiawithselectiveintubation,9awakeepiduralanaesthesia and 21 spontaneous breathing anaesthesia with laryngeal mask. The mean operation time was 31 min (SD: 10.2). The median postoperative drainage period and hospital stay were 2 days (range of 1–11) and 3 days (range of 2–11), respectively. Prolonged air leak occurredin 1patient (1.4%).Overamean follow-upperiod of 60 months (SD: 22.5), tworecurrences (2.7%)were reported. CONCLUSIONS: The transcollation® technique by cold coagulation of blebs and bullae seems to be effective in the treatment of primary spontaneous pneumothorax. Owing to its potential advantages, it appears to be particularly suitable to be associated with awake epidural and LMA anaesthesia