Activation of KLF1 Enhances the Differentiation and Maturation of Red Blood Cells from Human Pluripotent Stem Cells.

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.

Human induced pluripotent stem cell derived erythroblasts can undergo definitive erythropoiesis and co-express gamma and beta globins.

Human induced pluripotent stem cells (hiPSCs), like embryonic stem cells, are under intense investigation for novel approaches to model disease and for regenerative therapies. Here, we describe the derivation and characterization of hiPSCs from a variety of sources and show that, irrespective of origin or method of reprogramming, hiPSCs can be differentiated on OP9 stroma towards a multi-lineage haemo-endothelial progenitor that can contribute to CD144(+) endothelium, CD235a(+) erythrocytes (myeloid lineage) and CD19(+) B lymphocytes (lymphoid lineage). Within the erythroblast lineage, we were able to demonstrate by single cell analysis (flow cytometry), that hiPSC-derived erythroblasts express alpha globin as previously described, and that a sub-population of these erythroblasts also express haemoglobin F (HbF), indicative of fetal definitive erythropoiesis. More notably however, we were able to demonstrate that a small sub-fraction of HbF positive erythroblasts co-expressed HbA in a highly heterogeneous manner, but analogous to cord blood-derived erythroblasts when cultured using similar methods. Moreover, the HbA expressing erythroblast population could be greatly enhanced (44·0 ± 6·04%) when a defined serum-free approach was employed to isolate a CD31(+) CD45(+) erythro-myeloid progenitor. These findings demonstrate that hiPSCs may represent a useful alternative to standard sources of erythrocytes (RBCs) for future applications in transfusion medicine.

Human induced pluripotent stem cells are capable of B-cell lymphopoiesis.

Induced pluripotent stem (iPS) cells offer a unique potential for understanding the molecular basis of disease and development. Here we have generated several human iPS cell lines, and we describe their pluripotent phenotype and ability to differentiate into erythroid cells, monocytes, and endothelial cells. More significantly, however, when these iPS cells were differentiated under conditions that promote lympho-hematopoiesis from human embryonic stem cells, we observed the formation of pre-B cells. These cells were CD45(+)CD19(+)CD10(+) and were positive for transcripts Pax5, IL7αR, λ-like, and VpreB receptor. Although they were negative for surface IgM and CD5 expression, iPS-derived CD45(+)CD19(+) cells also exhibited multiple genomic D-J(H) rearrangements, which supports a pre-B-cell identity. We therefore have been able to demonstrate, for the first time, that human iPS cells are able to undergo hematopoiesis that contributes to the B-cell lymphoid lineage.

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

Red blood cells mature within the erythroblastic island (EI) niche that consists of specialized macrophages surrounded by differentiating erythroblasts. Here we establish an in vitro system to model the human EI niche using macrophages that are derived from human induced pluripotent stem cells (iPSCs), and are also genetically programmed to an EI-like phenotype by inducible activation of the transcription factor, KLF1. These EI-like macrophages increase the production of mature, enucleated erythroid cells from umbilical cord blood derived CD34+ haematopoietic progenitor cells and iPSCs; this enhanced production is partially retained even when the contact between progenitor cells and macrophages is inhibited, suggesting that KLF1-induced secreted proteins may be involved in this enhancement. Lastly, we find that the addition of three secreted factors, ANGPTL7, IL-33 and SERPINB2, significantly enhances the production of mature enucleated red blood cells. Our study thus contributes to the ultimate goal of replacing blood transfusion with a manufactured product.

Diagnostic performance of anti-cyclic citrullinated peptide and rheumatoid factor in patients with rheumatoid arthritis.

AIM: To compare the diagnostic performance of rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibody (anti-CCP) in the diagnosis of patients with rheumatoid arthritis (RA) in Taiwan. METHODS: Serum concentrations of RF and anti-CCP were measured in 246 cases, including 39 patients with RA and 207 patients with other rheumatic diseases (non-RA). The age, sex, clinical presentation, RF, anti-CCP results and the final diagnoses were recorded and analyzed. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (LR+) and negative likelihood ratio (LR-) were calculated. RESULTS: Among all 246 patients, 39 (15.9%) were diagnosed with RA and 207 (84.1%) were diagnosed with other rheumatic diseases (non-RA). In the diagnosis of RA, the sensitivity, specificity, PPV, NPV, LR+ and LR- of the RF test were 67%, 79%, 37%, 93%, 3.12, and 0.42, respectively. The corresponding data for the anti-CCP test were 79%, 98%, 86%, 96%, 32.91 and 0.21, respectively. The presence of either anti-CCP or RF increased the sensitivity to 85%, and when they both were present, the specificity increased to 98%. Among the 39 RA patients, 26 (66.7%) tested positive for RF, and 31 (79.5%) tested positive for anti-CCP. RF was positive in two of eight anti-CCP-negative patients with RA, and anti-CCP was positive in seven of 13 RF-negative patients with RA. CONCLUSIONS: The RF and anti-CCP tests are complementary, and the co-detection of these antibodies can increase the detection rate and provide important clinical value in the diagnosis of RA. Both anti-CCP and RF positivity are useful for the diagnosis of RA, and use of both tests together improves the diagnostic sensitivity.

Efficacy of Traditional Chinese Medicine in Xerostomia and Quality of Life during Radiotherapy for Head and Neck Cancer: A Prospective Pilot Study.

Xerostomia is one of the most common acute and late complications of radiotherapy for head and neck cancer, and it affects quality of life. We conducted a prospective study to evaluate the efficacy of traditional Chinese medicine (TCM) in toxicities and quality of life during radiotherapy. Head and neck cancer patients who were scheduled for radiotherapy were checked for inclusion/exclusion criteria before enrollment. Patients in the study group (inpatients) were hospitalized in a Chinese medicine ward and received concomitant TCM intervention during radiotherapy, while those in the control group (outpatients) received only conventional cancer treatments at the Western outpatient department. The primary end point was amelioration of postradiotherapy side effects. The secondary end points were quality of life during the cancer therapy and occurrence of adverse events following the TCM treatments. Thirty inpatients and 50 outpatients completed the study. Compared to the control group, those in the TCM group had decreased severity of xerostomia. There was no treatment-related impairment of renal or hepatic function among TCM group. Although better outcomes of social contact, dyspnea, physical and emotional function, and financial problems were found in the TCM group, we need further confirmation about the impact of hospitalization itself on these results.

Human induced pluripotent stem cell-derived B lymphocytes express sIgM and can be generated via a hemogenic endothelium intermediate.

The differentiation of human pluripotent stem cells to the B-cell lymphoid lineage has important clinical applications that include in vitro modeling of developmental lymphogenesis in health and disease. Here, we first demonstrate the capacity of human induced pluripotent stem cells (hiPSCs) to differentiate into CD144(+)CD73(-)CD43/CD235a(-) cells, characterized as hemogenic endothelium, and show that this population is capable of differentiating to CD10(+)CD19(+) B lymphocytes. We also demonstrate that B lymphocytes generated from hiPSCs are able to undergo full VDJ rearrangement and express surface IgM (sIgM(+)), thus representing an immature B-cell subset. Efficiency of sIgM expression on the hiPSC-derived B lymphocytes (∼ 5% of CD19(+) cells) was comparable with B lymphocytes generated from human umbilical cord blood (UCB) hematopoietic progenitor cells. Importantly, when assessed by global transcriptional profiling, hiPSC-derived B-cells show a very high level of similarity when compared with their UCB-derived counterparts, such that from more than 47,000 different transcripts, only 45 were significantly different (with a criteria adjusted P value P<0.05, log FC >1.5 or 2.8-fold). This represents a unique in vitro model to delineate critical events during lymphogeneisis in development and lymphoid diseases such as acute lymphocytic leukemia.

Discontinuation of anti-TNF-α therapy in a Chinese cohort of patients with rheumatoid arthritis.

The aim of this retrospective study was to examine the predictors of discontinuation of anti-tumor necrosis factor (TNF) therapy due to adverse events in Chinese patients with rheumatoid arthritis (RA). Anti-TNF-related adverse events were recorded and analyzed in 217 consecutive patients with RA followed in our institution from 2003 to 2010. Time to discontinuation of anti-TNF-α therapy was estimated using survival analysis techniques. The anti-TNF agents administered were etanercept in 181 patients and adalimumab in 36 patients. The mean age at diagnosis was 45.2 ± 13.5 years, and mean age at initiation of anti-TNF therapy was 51.8 ± 13.0 years. The mean duration of anti-TNF agent use was 36.0 ± 26.5 months (range, 1.4-87.0; median, 26.4 months). Of the 217 patients, 39 (18.0 %) developed adverse events [etanercept in 34 (18.8 %] and adalimumab in 5 (13.9 %)] during the treatment period (tuberculosis in 5, bacterial infections in 19, virus infection in 7, neuropathy in 3, malignancy in 3, other drug-related events in 1, and appendicitis in 1). In patients with RA, older age (≥55 years) at initiation of anti-TNF therapy [odds ratio (OR), 3.20; 95 % confidence interval (CI), 1.67-6.20; p < 0.001], Cr ≥1.5 mg/dL (OR, 5.72; 95 % CI, 1.17-27.90; p = 0.031), and occurrence of adverse events (OR, 3.82; 95 % CI, 1.75-8.35; p = 0.001) were associated with increased likelihood of discontinuation of anti-TNF treatment. In the present study, a significant proportion (7.8 %, 17/217) of patients with RA discontinued anti-TNF treatment because of adverse events. In the elderly and in patients with renal insufficiency, caution is needed when starting anti-TNF treatment.

Efficient differentiation of human induced pluripotent stem cells generates cardiac cells that provide protection following myocardial infarction in the rat.

Induced pluripotent stem (iPS) cells are being used increasingly to complement their embryonic counterparts to understand and develop the therapeutic potential of pluripotent cells. Our objectives were to identify an efficient cardiac differentiation protocol for human iPS cells as monolayers, and demonstrate that the resulting cardiac progenitors could provide a therapeutic benefit in a rodent model of myocardial infarction. Herein, we describe a 14-day protocol for efficient cardiac differentiation of human iPS cells as a monolayer, which routinely yielded a mixed population in which over 50% were cardiomyocytes, endothelium, or smooth muscle cells. When differentiating, cardiac progenitors from day 6 of this protocol were injected into the peri-infarct region of the rat heart; after coronary artery ligation and reperfusion, we were able to show that human iPS cell-derived cardiac progenitor cells engrafted, differentiated into cardiomyocytes and smooth muscle, and persisted for at least 10 weeks postinfarct. Hearts injected with iPS-derived cells showed a nonsignificant trend toward protection from decline in function after myocardial infarction, as assessed by magnetic resonance imaging at 10 weeks, such that the ejection fraction at 10 weeks in iPS treated hearts was 62%±4%, compared to that of control infarcted hearts at 45%±9% (P<0.2). In conclusion, we demonstrated efficient cardiac differentiation of human iPS cells that gave rise to progenitors that were retained within the infarcted rat heart, and reduced remodeling of the heart after ischemic damage.

Interactions between beta subunits of the KCNMB family and Slo3: beta4 selectively modulates Slo3 expression and function.

BACKGROUND: The pH and voltage-regulated Slo3 K(+) channel, a homologue of the Ca(2+)- and voltage-regulated Slo1 K(+) channel, is thought to be primarily expressed in sperm, but the properties of Slo3 studied in heterologous systems differ somewhat from the native sperm KSper pH-regulated current. There is the possibility that critical partners that regulate Slo3 function remain unidentified. The extensive amino acid identity between Slo3 and Slo1 suggests that auxiliary beta subunits regulating Slo1 channels might coassemble with and modulate Slo3 channels. Four distinct beta subunits composing the KCNMB family are known to regulate the function and expression of Slo1 Channels. METHODOLOGY/PRINCIPAL FINDINGS: To examine the ability of the KCNMB family of auxiliary beta subunits to regulate Slo3 function, we co-expressed Slo3 and each beta subunit in heterologous expression systems and investigated the functional consequences by electrophysiological and biochemical analyses. The beta4 subunit produced an 8-10 fold enhancement of Slo3 current expression in Xenopus oocytes and a similar enhancement of Slo3 surface expression as monitored by YFP-tagged Slo3 or biotin labeled Slo3. Neither beta1, beta2, nor beta3 mimicked the ability of beta4 to increase surface expression, although biochemical tests suggested that all four beta subunits are competent to coassemble with Slo3. Fluorescence microscopy from beta4 KO mice, in which an eGFP tag replaced the deleted exon, revealed that beta4 gene promoter is active in spermatocytes. Furthermore, quantitative RT-PCR demonstrated that beta4 and Slo3 exhibit comparable mRNA abundance in both testes and sperm. CONCLUSIONS/SIGNIFICANCE: These results argue that, for native mouse Slo3 channels, the beta4 subunit must be considered as a potential interaction partner and, furthermore, that KCNMB subunits may have functions unrelated to regulation of the Slo1 alpha subunit.