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

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

SIGLEAvailable from British Library Document Supply Centre- DSC:DXN065235 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

High efficiency serum free feeder free erythroid differentiation of human pluripotent stem cells using small molecules

This article describes a good manufacturing practice (GMP)-compatible, feeder-free and serum-free method to produce large numbers of erythroid cells from human pluripotent stem cells (hPSCs), either embryonic or induced. This multistep protocol combines cytokines and small molecules to mimic and surpass the early stages of development. It produces, without any selection or sorting step, a population of cells in which 91.8% ± 5.4% express CD34 at day 7, 98.6% ± 1.3% express CD43 at day 10, and 99.1% ± 0.95% of cells are CD235a positive by day 31 of the differentiation process. Moreover, this differentiation protocol supports extensive expansion, with a single hPSC producing up to 150 hematopoietic progenitor cells by day 10 and 50,000–200,000 erythroid cells by day 31. The erythroid cells produced exhibit a definitive fetal hematopoietic type, with 90%–95% fetal globin and variable proportion of embryonic and adult globin at the protein level. The presence of small molecules during the differentiation protocol has quantitative and qualitative effects; it increases the proportion of adult globin and decreases the proportion of embryonic globin. Given its level of definition, this system provides a powerful tool for investigation of the mechanisms governing early hematopoiesis and erythropoiesis, including globin switching and enucleation. The early stages of the differentiation protocol could also serve as a starting point for the production of endothelial cells and other hematopoietic cells, or to investigate the production of long-term reconstituting hematopoietic stem cells from hPSCs

A stable analogue of glucose-dependent insulinotropic polypeptide, GIP(LysPAL16), enhances functional differentiation of mouse embryonic stem cells into cells expressing islet-specific genes and hormones

Embryonic stem (ES) cells can be differentiated into insulin-producing cells by conditioning the culture media. However, the number of insulin-expressing cells and amount of insulin released is very low. Glucose-dependent insulinotropic polypeptide (GIP) enhances the growth and differentiation of pancreatic beta-cells. This study examined the potential of the stable analogue GIP(LysPAL16) to enhance the differentiation of mouse ES cells into insulin-producing cells using a five-stage culturing strategy. Semi-quantitative PCR indicated mRNA expression of islet development markers (nestin, Pdx1, Nkx6.1, Oct4), mature pancreatic beta-cell markers (insulin, glucagon, Glut2, Sur1, Kir6.1) and the GIP receptor gene GIP-R in undifferentiated (stage 1) cells, with increasing levels in differentiated stages 4 and 5. IAPP and somatostatin genes were only expressed in differentiated stages. Immunohistochemical studies confirmed the presence of insulin, glucagon, somatostatin and IAPP in differentiated ES cells. After supplementation with GIP(LysPAL16), ES cells at stage 4 released insulin in response to secretagogues and glucose in a concentration-dependent manner, with 35-100% increases in insulin release. Cellular C-peptide content also increased by 45% at stages 4 and 5. We conclude that the stable GIP analogue enhanced differentiation of mouse ES cells towards a phenotype expressing specific beta-cell genes and releasing insulin.sch_die387pub4493pub

Umweltfreundliche Stueckverzinkung Schlussbericht

Available from TIB Hannover: FR 5978+a / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman

Large-scale transcriptional profiling and functional assays reveal important roles for Rho-GTPase signalling and SCL during haematopoietic differentiation of human embryonic stem cells

nderstanding the transcriptional cues that direct differentiation of human embryonic stem cells (hESCs) and human-induced pluripotent stem cells to defined and functional cell types is essential for future clinical applications. In this study, we have compared transcriptional profiles of haematopoietic progenitors derived from hESCs at various developmental stages of a feeder- and serum-free differentiation method and show that the largest transcriptional changes occur during the first 4 days of differentiation. Data mining on the basis of molecular function revealed Rho-GTPase signalling as a key regulator of differentiation. Inhibition of this pathway resulted in a significant reduction in the numbers of emerging haematopoietic progenitors throughout the differentiation window, thereby uncovering a previously unappreciated role for Rho-GTPase signalling during human haematopoietic development. Our analysis indicated that SCL was the 11th most upregulated transcript during the first 4 days of the hESC differentiation process. Overexpression of SCL in hESCs promoted differentiation to meso-endodermal lineages, the emergence of haematopoietic and erythro-megakaryocytic progenitors and accelerated erythroid differentiation. Importantly, intrasplenic transplantation of SCL-overexpressing hESC-derived haematopoietic cells enhanced recovery from induced acute anaemia without significant cell engraftment, suggesting a paracrine-mediated effect