Manipulating transcription factors in human induced pluripotent cell-derived cells to enhance the production and the maturation of red blood cells

The most widely transfused blood component is red blood cells (RBCs), and voluntary donation is the main resource for RBC transfusion. In the UK, 7,000 units of RBCs are transfused daily but this life-saving cell therapy is completely dependent on donors and there are persistent problems associated with transfusion transmitted infections and in blood group compatibility. Furthermore, the quality, safety and efficiency of donated RBCs gradually decrease with storage time. A number of novel sources of RBCs are being explored including the production of RBCs from adult haematopoietic progenitor cells, erythroid progenitor cell lines and induced pluripotent stem cells (iPSCs). The iPSC source could essentially provide a limitless supply and a route to producing cells that are matched to the recipient. A number of protocols have been described to produce mature RBCs from human pluripotent stem cells but they are relatively inefficient and would be difficult to scale up to the levels required for clinical translation. We tested and evaluated a defined feeder- and serum-free differentiation protocol for deriving erythroid cells from hiPSCs. RBC production was not efficient, the cells that were produced did not enucleate efficiently and they expressed embryonic rather than adult globin. We hypothesised that the production of RBCs from iPSCs could be enhanced by enforced expression of erythroid-specific transcription factors (TFs). Previous studies had demonstrated that Krüppel-like factor 1 (KLF1) plays an important role in RBC development and maturation so we generated iPSC lines expressing a tamoxifen-inducible KLF1-ERT2 fusion protein. Using zinc finger nuclease technology, we targeted the expression cassette to the AAVS1 locus to ensure consistent expression levels and to avoid integration site specific effects and/or silencing. These iKLF1 iPSCs were applied to our defined RBC differentiation protocol and the activity of KLF1 was induced by adding tamoxifen. Activation of KLF1 from day 10 accelerated erythroid differentiation and maturation with an increase in the proportion of erythroblasts, a higher level of expression of erythroid genes associated with maturation and an apparently more robust morphology. However, KLF1 activation had an anti-proliferation effect resulting in significantly less cell generated overall and HPLC analysis demonstrated that KLF1-activated cells expressed higher levels of embryonic globin compared to control iPSCs-derived cells. Many of the effects that were observed when KLF1 was activated from day 10 were not observed when activated from day 18. We therefore concluded that activation of exogenous KLF1 is able to promote erythroid cell production and maturation in progenitors (day 10) but not at the later stage of erythropoiesis (day 18). We hypothesised that KLF1 might require a co-factor to regulate RBC maturation and adult globin expression at the later stage of erythropoiesis. The TF, B-cell lymphoma/leukaemia 11a (BCL11A), plays a key role in the suppression of foetal globin expression, thereby completing globin switching to adult globin. Preliminary data showed that iPSC-derived erythroid cells were able to express adult globin when transduced with a BCL11A-expressing lentiviral-vector. Based on that finding we then generated an iPSC line expressing tamoxifen-inducible BCL11AERT2 and KLF1-ERT2 fusion proteins, applied this iBK iPSC line to our differentiation protocol. Activation of both TFs from day 18 slightly increased the expression of genes associated with RBC maturation and the inclusion of BCL11A appeared to eliminate the anti-proliferation effect of KLF1. Most importantly, activation of both BCL11A and KLF1 from day 18 of the differentiation protocol increased the production of α- globin (foetal / adult globin) indicating that some definitive-like erythroid cells might be generated by activation of both TFs at the later stage of erythroid differentiation. Collectively, these findings demonstrate that enforced expression of erythroid TFs could be a useful strategy to enhance RBC maturation from iPSCs

Enforced Expression of HOXB4 in Human Embryonic Stem Cells Enhances the Production of Hematopoietic Progenitors but Has No Effect on the Maturation of Red Blood Cells

We have developed a robust, Good Manufacturing Practice-compatible differentiation protocol capable of producing scalable quantities of red blood cells (RBCs) from human pluripotent stem cells (hPSCs). However, translation of this protocol to the clinic has been compromised because the RBCs produced are not fully mature; thus, they express embryonic and fetal, rather than adult globins, and they do not enucleate efficiently. Based on previous studies, we predicted that activation of exogenous HOXB4 would increase the production of hematopoietic progenitor cells (HPCs) from hPSCs and hypothesized that it might also promote the production of more mature, definitive RBCs. Using a tamoxifen-inducible HOXB4-ERT2 expression system, we first demonstrated that activation of HOXB4 does increase the production of HPCs from hPSCs as determined by colony-forming unit culture activity and the presence of CD43+CD34+ progenitors. Activation of HOXB4 caused a modest, but significant, increase in the proportion of immature CD235a+/CD71+ erythroid cells. However, this did not result in a significant increase in more mature CD235a+/CD71− cells. RBCs produced in the presence of enhanced HOXB4 activity expressed embryonic (ε) and fetal (γ) but not adult (β) globins, and the proportion of enucleated cells was comparable to that of the control cultures. We conclude that programming with the transcription factor HOXB4 increases the production of hematopoietic progenitors and immature erythroid cells but does not resolve the inherent challenges associated with the production of mature adult-like enucleated RBCs

Fractures in sub-Saharan Africa: epidemiology, economic impact and ethnography (Fractures-E3): study protocol

Background: The population of older adults is growing in sub-Saharan Africa. Ageing exponentially increases fragility fracture risk. Of all global regions, Africa is projected to observe the greatest increase in fragility fractures. Fractures cause pain, disability and sometimes death, and management is expensive, often requiring complex healthcare delivery. For countries to plan future healthcare services, understanding is needed of fracture epidemiology, associated health service costs and the currently available healthcare resources. Methods: The Fractures-E3 5-year mixed-methods research programme will investigate the epidemiology, economic impact, and treatment provision for fracture and wider musculoskeletal health in The Gambia, South Africa and Zimbabwe. These three countries are diverse in their geography, degree of urbanisation, maturity of health service infrastructure, and health profiles. The programme comprises five study types: (i) population-based cross-sectional studies to determine vertebral fracture prevalence. Secondary outcomes will include osteoarthritis and sarcopenia. Age- and sex-stratified household sampling will recruit 5030 adults aged 40 years and older; (ii) prospective cohort studies in adults aged 40 years and older will determine hip fracture incidence, associated risk factors, and outcomes over one year (e.g. mortality, disability, health-related quality of life); (iii) economic studies of direct health costs of hip fracture with projection modelling of future national health costs and cost-effectiveness analyses of different hip fracture care pathways; (iv) national surveys of hip fracture services (including traditional bonesetters in The Gambia); and (v) ethnographic studies of hip fracture care provision and experiences will understand fracture service pathways. Conclusions: Greater understanding of current and expected fracture burdens, fracture risk factors, and existing fracture care provision, is intended to inform national clinical guidelines, health service policy and planning and future health service development in sub-Saharan Africa.</ns4:p

Novel peptide discovery Isolation and characterisation of peptides with antidiabetic properties from the skin secretions of amphibians

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