With increasing worldwide demand for safe blood, there is much interest in generating red blood cells in vitro as an alternative clinical product. However, available methods for in vitro generation of red cells from adult and cord blood progenitors do not yet provide a sustainable supply, and current systems using pluripotent stem cells as progenitors do not generate viable red cells. We have taken an alternative approach, immortalizing early adult erythroblasts generating a stable line, which provides a continuous supply of red cells. The immortalized cells differentiate efficiently into mature, functional reticulocytes that can be isolated by filtration. Extensive characterization has not revealed any differences between these reticulocytes and in vitro-cultured adult reticulocytes functionally or at the molecular level, and importantly no aberrant protein expression. We demonstrate a feasible approach to the manufacture of red cells for clinical use from in vitro culture.

Cord blood stem cells are an attractive starting source for the production of red blood cells in vitro for therapy because of additional expansion potential compared with adult peripheral blood progenitors and cord blood banks usually being more representative of national populations than blood donors. Consequently, it is important to establish how similar cord RBCs are to adult cells. In this study, we used multiplex tandem mass tag labeling combined with nano-LC-MS/MS to compare the proteome of adult and cord RBCs and reticulocytes. 2838 unique proteins were identified, providing the most comprehensive compendium of RBC proteins to date. Using stringent criteria, 1674 proteins were quantified, and only a small number differed in amount between adult and cord RBC. We focused on proteins critical for RBC function. Of these, only the expected differences in globin subunits, along with higher levels of carbonic anhydrase 1 and 2 and aquaporin-1 in adult RBCs would be expected to have a phenotypic effect since they are associated with the differences in gaseous exchange between adults and neonates. Since the RBC and reticulocyte samples used were autologous, we catalogue the change in proteome following reticulocyte maturation. The majority of proteins (>60% of the 1671 quantified) reduced in abundance between 2-and 100-fold following maturation. However, ϳ5% were at a higher level in RBCs, localized almost exclusively to cell membranes, in keeping with the known clearance of intracellular recycling pools during reticulocyte maturation. Overall, these data suggest that, with respect to the proteome, there is no barrier to the use of cord progenitors for the in vitro generation of RBCs for transfusion to adults other than the expression of fetal, not adult, hemoglobin. Molecular & Cellular Proteomics

Blood transfusion is widely used in the clinic but the source of red blood cells (RBCs) is dependent on donors, procedures are susceptible to transfusion‐transmitted infections and complications can arise from immunological incompatibility. Clinically‐compatible and scalable protocols that allow the production of RBCs from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have been described but progress to translation has been hampered by poor maturation and fragility of the resultant cells. Genetic programming using transcription factors has been used to drive lineage determination and differentiation so we used this approach to assess whether exogenous expression of the Erythroid Krüppel‐like factor 1 (EKLF/KLF1) could augment the differentiation and stability of iPSC‐derived RBCs. To activate KLF1 at defined time points during later stages of the differentiation process and to avoid transgene silencing that is commonly observed in differentiating pluripotent stem cells, we targeted a tamoxifen‐inducible KLF1‐ERT2 expression cassette into the AAVS1 locus. Activation of KLF1 at day 10 of the differentiation process when hematopoietic progenitor cells were present, enhanced erythroid commitment and differentiation. Continued culture resulted the appearance of more enucleated cells when KLF1 was activated which is possibly due to their more robust morphology. Globin profiling indicated that these conditions produced embryonic‐like erythroid cells. This study demonstrates the successful use of an inducible genetic programing strategy that could be applied to the production of many other cell lineages from human induced pluripotent stem cells with the integration of programming factors into the AAVS1 locus providing a safer and more reproducible route to the clinic. Stem Cells 2017;35:886–897

The peptide oxytocin is present in tissues of the male reproductive tract from a variety of mammalian species. In the human, specific mRNA for oxytocin and the peptide itself have been identified in the testis, epididymis and prostate. The peptide has been shown to modulate both steroidogenesis and contractility in the male reproductive tract and may be involved in the pathogenesis of benign prostatic hyperplasia. We have performed Western blots and immunohistochemistry using a specific antibody to the human oxytocin receptor (OTR) to investigate the distribution and localization of the receptor in the human and macaque monkey (Macaca fasicularis). An immunoreactive band of approximately 55 kDa was detected in human and monkey uterine, testicular and prostatic tissues and in preparations of monkey caput and cauda epididymis. A second, less intense, band of 60 kDa was also seen in testicular and uterine tissue samples. No specific bands were detected in monkey muscle or in any tissue following incubation with mouse immunoglobulin (Ig)M. In the human and monkey testis staining for the OTR was present in the interstitial tissue and in Sertoli cells. Localization of the OTRs varied throughout the epididymis being expressed by epithelial cells proximally but confined to cells at the base of the epididymal ducts and to the surrounding smooth muscle layers distally. In the prostate OTR were localized to the stromal tissue surrounding the ducts. These findings correlate with sites of local production of the peptide and the observed biological actions of oxytocin, and thus support the evidence that oxytocin may play a physiological role in the male reproductive tract.

Induced pluripotent stem cells (iPSC) are an attractive progenitor source for the generation of in vitro blood products. However, before iPSC-derived erythroid cells can be considered for therapeutic use their similarity to adult erythroid cells must be confirmed. We have analysed the proteome of erythroid cells differentiated from the iPSC fibroblast derived line (C19) and showed they express hallmark RBC proteins, including all those of the ankyrin and 4.1R complex. We next compared the proteome of erythroid cells differentiated from three iPSC lines (C19, OCE1, OPM2) with that of adult and cord blood progenitors. Of the 1989 proteins quantified <3% differed in level by 2-fold or more between the different iPSC-derived erythroid cells. When compared to adult cells, 11% of proteins differed in level by 2-fold or more, falling to 1.9% if a 5-fold threshold was imposed to accommodate slight inter-cell line erythropoietic developmental variation. Notably, the level of >30 hallmark erythroid proteins was consistent between the iPSC lines and adult cells. In addition, a sub-population (10–15%) of iPSC erythroid cells in each of the iPSC lines completed enucleation. Aberrant expression of some cytoskeleton proteins may contribute to the failure of the majority of the cells to enucleate since we detected some alterations in cytoskeletal protein abundance. In conclusion, the proteome of erythroid cells differentiated from iPSC lines is very similar to that of normal adult erythroid cells, but further work to improve the induction of erythroid cells in existing iPSC lines or to generate novel erythroid cell lines is required before iPSC-derived red cells can be considered suitable for transfusion therapy.