Synergy between erythropoietin and stem cell factor during erythropoiesis can be quantitatively described without co-signaling effects

Synergistic interactions between cytokines underlie developmental processes fundamental to tissue and cellular engineering. However, a mechanistic understanding of the cell-specific and population-mediated effects is often lacking. In this study, we have investigated the synergistic generation of erythroid cells in response to erythropoietin (EPO) and stem cell factor (SCF). We have used a quantitative approach to determine if the effects of EPO and SCF superpose in a supra-additive fashion on the cell proliferation rate or on the death rate, suggesting a contribution from a joint cytokine effect (co-signaling). Primary mouse bone marrow hematopoietic cells and the stem cell-like FDCP-mix cell line were used to investigate the effects of EPO and SCF (individually or in combination) on erythroid output. Carboxyfluorescein diacetate succinimidyl ester (CFSE)-based cell-division tracking and mathematical modeling were used to measure cell type-specific proliferation and death rates. We observed a significant synergistic effect of EPO and SCF on the net generation of benzidine positive (erythroid) colony-forming cells, CD71++ (early erythroblasts) cells and TER-119+ (late erythroblasts and reticulocytes) cells in culture. When the observed increases in cell number were decomposed into proliferation and death rates, the cytokines were shown to act independently at different stages of erythroid development; SCF promoted the early proliferation of primitive cells, while EPO primarily promoted the survival of differentiating erythroid progenitor cells. Our analysis demonstrates that EPO and SCF have distinct and predominantly sequential effects on erythroid differentiation. This study emphasizes the necessity to separate proliferation rates from death rates to understand apparent cytokine synergies.     

c-myc expression affects proliferation but not terminal differentiation or survival of explanted erythroid progenitor cells

The expression of c-myc was analyzed in murine and human erythroblasts throughout their differentiation in vitro into reticulocytes. The murine cells were splenic erythroblasts from animals infected with the anemia strain of Friend virus (FVA cells). In FVA cells cultured without EPO, the c-myc mRNA and protein levels decrease sharply within 3 to 4 h, showing that continual EPO stimulation is required to maintain c-myc expression. When cultured with EPO, the c-myc mRNA level of FVA cells is raised within 30 min of exposure. The c-myc mRNA and protein reach maxima at 1 to 3 h, then decline slowly to very low levels by 18 h. In contrast, c-fos and c-jun mRNA levels are not regulated by EPO in FVA cells. The human cells analyzed were colony-forming units-erythroid, CFU-E, derived in vitro by the culture of peripheral blood burst-forming units-erythroid (BFU-E). When grown in EPO and insulin-like growth factor 1 (IGF-1) these cells differentiate into reticulocytes over 6 days rather than the 2 days required for murine cells, but the c-myc mRNA kinetics and response to EPO parallel those of mouse cells at similar stages of differentiation. Both IGF-1 and c-kit ligand (SCF) cause an additive increase in c-myc mRNA in human CFU-E in conjunction with EPO. These additive effects suggest that EPO, IGF-1, and SCF affect c-myc mRNA accumulation by distinct mechanisms. Addition of an antisense oligonucleotide to c-myc in cultures of human CFU-E specifically inhibited cell proliferation but did not affect erythroid cell differentiation or apoptosis. When human cells were grown in high SCF concentrations, an environment which enhances proliferation and retards differentiation, antisense oligonucleotide to c-myc strongly inhibited proliferation, but such inhibition did not induce differentiation. This latter result indicates that differentiation requires signals other than depression of c-myc and resultant depression of proliferation. © 1996 Wiley-Liss, Inc.     

Induction of circulating neonatal stem cell populations

Hematopoietic cell differentiation and growth are regulated by paracrine molecules that include insulin and insulin-like growth factors (IGFs). IGF-I and -II stimulation of erythropoiesis in cultures of adult bone marrow and peripheral blood cells and murine fetal liver cells has been previously reported. In order to investigate whether these paracrines also influence differentiation and proliferation of human neonatal progenitor cells, we assessed their effects in cultures of umbilical cord blood and adult blood and marrow cells, using a serum-substituted system. IGF-I stimulated colony-forming unit-erythroid (CFU-E)-derived colony formation by adult cells by up to 265%, while IGF-II augmented colony formation by up to 100% in the presence of erythropoietin. Stimulation occurred in a saturable fashion over concentrations of 0 to 200 ng/ml. Similar results were obtained in subcultures of adult-circulating progenitors. Moreover, a subpopulation of erythropoietin-independent adult CFU-E was stimulated to proliferate by IGF-I but not by IGF-II. In contrast to these effects in adult marrow culture, IGF-II exerted a greater stimulatory effect on neonatal CFU-E proliferation than did IGF-I in erythropoietin-containing cultures. Additionally, IGF-II stimulated proliferation of erythropoietin-independent neonatal CFU-Es in a concentration-dependent fashion. Together, the data are consistent with the hypothesis that somatomedins are involved in developmental regulation of erythropoiesis.     

CFU-E和其分化细胞的生物物理特性

用可诱发小鼠贫血的病毒(anemia-inducing strain friend’s virus,FVA)感染BALB／c小鼠,13d后取其脾脏,用Ficoll-Urografin分层液(1．070)分离出红系集落形成单位(colony forming unit-etythroid,CFU-E)细胞,用Wright-Giemsa染液染色并用透射电镜进行形态学观察,在培养液加细胞因子,如促红细胞生成素(erythropoietin,EPO)、白介素3(interleukin-3,IL-3)、干细胞刺激因子(stem cell factor,SCF),诱导其分化的情况下培养12、24和36h,分别对0、12、24和36h的细胞进行电泳率、膜的流动性和变形性的测量及红系特异转录因子GATA-1表达(0、12、24和48h)的检测,发现CFU-E细胞随着分化培养时间增加,其电泳率不断减少,膜的流动性不断增大,细胞的变形性和取向性逐渐变好;CFU-E在0、12和24hGATA持续高水平表达,而48h后,其表达明显降低．     

Murine pluripotent hematopoietic progenitors constitutively expressing a normal erythropoietin receptor proliferate in response to erythropoietin without preferential erythroid cell differentiation.

Erythropoietin (EPO) is a prime regulator of the growth and differentiation of erythroid blood cells. The EPO receptor (EPO-R) is expressed in late erythroid progenitors (mature BFU-E and CFU-E), and EPO induces proliferation and differentiation of these cells. By introducing, with a retroviral vector, a normal EPO-R cDNA into murine adult bone marrow cells, we showed that EPO is also able to induce proliferation in pluripotent progenitor cells. After 7 days of coculture with virus-producing cells, bone marrow cells were plated in methylcellulose culture in the presence of EPO, interleukin-3, or Steel factor alone or in combination. In the presence of EPO alone, EPO-R virus-infected bone marrow cells gave rise to mixed colonies comprising erythrocytes, granulocytes, macrophages and megakaryocytes. The addition of interleukin-3 or Steel factor to methylcellulose cultures containing EPO did not significantly modify the number of mixed colonies. The cells which generate these mixed colonies have a high proliferative potential as shown by the size and the ability of the mixed colonies to give rise to secondary colonies. Thus, it appears that EPO has the same effect on EPO-R-expressing multipotent cell proliferation as would a combination of several growth factors. Finally, our results demonstrate that inducing pluripotent progenitor cells to proliferate via the EPO signaling pathway has no major influence on their commitment.     

Evidence suggesting a stimulatory role for interleukin-10 in erythropoiesis in vitro

Interleukin-10 (IL-10) has been shown to exert anti-inflammatory effects by suppressing macrophage proliferation and inhibiting cytokine production. In this study we show that in the presence of erythropoietin (EPO), the addition of IL-10 results in a significant dose-dependent increase in both Burst Forming Unit-Erythroid (BFU-E) and Colony Forming Unit-Erythroid (CFU-E) colony growth in both serum-containing and serum-free murine cultures in vitro. IL-10 acts at the later stages of erythroid cell proliferation and differentiation as the increase in colony number was greater in CFU-E than in BFU-E, and was similar when IL-10 was added to BFU-E cultures at the time of culture initiation as when its addition to culture was delayed for 7 days. Furthermore, no increase in BFU-E colony number was noted when IL-10, added at the time of culture initiation, was neutralized by the addition to culture of a monoclonal anti-IL-10 antibody up to 7 days later. The increases in BFU-E by IL-10 addition were not the result of prolongation of BFU-E colony lifespan, which was not significantly different in IL-10 treated and control cultures, respectively. Rather IL-10 stimulated the proliferation of erythroid clusters that were now large enough to be recognized as colonies. IL-10-induced stimulation of erythropoiesis appeared to be independent of its inhibitory effects on macrophage function, as stimulation of erythroid colony growth was similar in macrophage-containing and depleted cultures. Studies to determine if the IL-10 effect was direct or indirect yielded equivocal results. A limiting dilution assay suggested a direct effect. However, a log/log dose response curve with IL-10 did not pass through the origin suggesting an indirect effect. These studies indicate that IL-10 acts synergistically with EPO to significantly increase stimulation of erythroid differentiation and proliferation in vitro and may be involved in the regulation of normal erythropoiesis in vivo. © 1996 Wiley-Liss, Inc.     

Elevation of Survivin levels by hematopoietic growth factors occurs in quiescent CD34+ hematopoietic stem and progenitor cells before cell cycle entry

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is overexpressed during G(2)/M phase in most cancer cells. In contrast, we previously reported that Survivin is expressed throughout the cell cycle in normal CD34(+) hematopoietic stem and progenitor cells stimulated by the combination of Thrombopoietin (Tpo), Stem Cell Factor (SCF) and Flt3 ligand (FL). In order to address whether Survivin expression is specifically up-regulated by hematopoietic growth factors before cell cycle entry, we isolated quiescent CD34(+) cells and investigated Survivin expression in response to growth factor stimulation. Survivin is up-regulated in CD34(+) cells with 2N DNA content following growth factor addition, suggesting it becomes elevated during G(0)/G(1). Survivin is barely detectable in freshly isolated umbilical cord blood (UCB) Ki-67(negative) and Cyclin D(negative) CD34(+) cells, however incubation with Tpo, SCF and FL for 20 hrs results in up-regulation without entry of cells into cell cycle. Culture of G(0) CD34(+) cells isolated based on Hoechst 33342/PyroninY staining with Tpo, SCF and FL for 48 hrs, results in significantly elevated Survivin mRNA and protein levels. Moreover, labeling of fresh G(0) CD34(+) cells with 5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) before culture with growth factors for up to 72 hrs, revealed that Survivin expression was elevated in CFSE(bright) G(0) CD34(+) cells, indicating that up-regulation occurred before entry into G1. These results suggest that up-regulation of Survivin expression in CD34(+) cells is an early event in cell cycle entry that is regulated by hematopoietic growth factors and does not simply reflect cell cycle progression and cell division.     

Developmental changes in erythropoietin receptor expression of fetal mouse liver.

Erythropoietin (EPO) stimulates proliferation and differentiation of late erythroid precursor cells (CFU-E) and thereby determines the rate of erythropoiesis. Liver is the major erythropoietic site in a fetus. We dealt with developmental changes in CFU-E and EPO receptor (EPO-R) of fetal mouse liver. The affinity of the EPO-R to EPO was unchanged during fetal development. The population size of CFU-E, the number of EPO-R per liver cell, and EPO-R mRNA decreased as gestation proceeded, in a pattern indicating that the expression of EPO-R on erythroid precursor cells in fetal mouse liver is governed mostly by the process of mRNA production.     

Distinct functions of erythropoietin and stem cell factor are linked to activation of mTOR kinase signaling pathway in human erythroid progenitors

Erythropoietin (EPO) and Stem Cell Factor (SCF) have partially distinct functions in erythroid cell development. The primary functions of EPO are to prevent apoptosis and promote differentiation, with a minor role as a mitogen. On the other hand SCF acts primarily as a mitogenic factor promoting erythroid cell proliferation with a minor role in inhibition of apoptosis. The concerted effects of these two growth factors are responsible for guiding initial commitment, expansion and differentiation of progenitors. The aim of the study was to identify signaling elements pertinent to translational control and elucidate whether both cytokines can contribute to protein translation providing some functional redundancy as seen with respect to apoptosis. The current study focused on non-apoptotic functions of SCF mediated through mTOR/p70S6 leading to protein translation and cell proliferation. We utilized a human primary erythroid progenitors and erythroblasts that are responsive to EPO and SCF to investigate the activation of mTOR/p70S6 kinases and their downstream effectors, the pathway primarily responsible for protein translation. We showed that mTOR, p70S6 kinases and their downstream signaling elements 4EBP1 and S6 ribosomal protein are all activated by SCF but not by EPO in primary erythroid progenitors. We also found that SCF is the sole contributor to activation of the protein translational machinery and activation of mTOR/p70S6 pathway is confined to the proliferative phase of erythroid differentiation program. Altogether these results demonstrate that unlike the survival function which is supported by both EPO and SCF protein translation essential for proliferation is governed by only SCF.     

Systematic experimental design for bioprocess characterization: Elucidating transient effects of multi-cytokine contributions on erythroid differentiation

In vitro differentiation of hematopoietic stem cells (HSCs) is a highly dynamic process whereby contributions of exogenous cytokines vary at each stage of differentiation. In this study, we present erythroid differentiation as three progressive yet independent stages and aim to elucidate transient contributions from stem cell factor (SCF), insulin-growth factor II (IGF-II), and erythropoietin (EPO). This will be accomplished using the Taguchi design and response surface methodology (RSM). We found that cultures with high process variability (noise factors), such as those in primary cell cultures, pose limitations on the effectiveness of RSM and result in inconsistencies in empirical models developed for elucidating transient effects. However, the Taguchi design—which showed greater robustness in accommodating for noise factors—successfully identified significant main and interactive contributions at each differentiation stage, thus highlighting the dynamic roles of each cytokine. The Taguchi analysis suggested high IGF-II dependency during early erythroid differentiation, with an antagonistic effect in the presence of EPO. At mid-stage differentiation, the roles of SCF and EPO dominate those of IGF-II, and the former act independently. Finally, toward erythroid maturation, only EPO plays a significant role. Although process outcomes from the Taguchi analysis were semi-quantitative, this approach provides a path for overcoming cell culture and sample-to-sample variability and can therefore be utilized with many cell culture applications in order to understandcomplex and intricate process relationships.     

Exercise-induced changes to in vitro T-lymphocyte mitogen responses using CFSE.

Carboxyfluorescein diacetate succinamidyl ester (CFSE) labeling of lymphocyte populations can provide unique insights into cell function at rest and with exercise, due to its ability to quantify cell division on an individual cell basis. This study aimed to characterize the effect of acute, intense exercise on T-lymphocyte function. Well-trained endurance runners completed 60 min of treadmill running at 95% of individual anaerobic threshold. Blood samples were collected before exercise; after 30 and 60 min of exercise; and after 30, 60, and 90 min of recovery. Isolated peripheral blood mononuclear cells were labeled with CFSE and cultured with or without mitogen (phytohemagglutinin). After culture, cell suspensions were labeled with CD3 (allophycocyanin) and CD8 (phycoerythrin), and expansion rates and cell death rates were calculated for each sample, as well as mitosis rates for each cell generation. Exercise was associated with a 60% decrease in cell expansion in both CD4 and CD8 cell types from before exercise to midexercise (P < 0.05). The significant decrease in expansion rate in the midexercise samples for both cell types was mirrored by a 65% increase in cell death (P < 0.05) in both cell types at that sample point. Exercise had no effect on the mitosis rate of either CD4 or CD8 cells in any cell generation (generations 0-3). This study indicates that 1 h of intense exercise affects in vitro T-lymphocyte function. These data suggest, for the first time, that exercise decreases cell expansion rate via an increase in cell death of both CD4 and CD8 T lymphocytes, rather than a decrease in mitosis.     

An in silico erythropoiesis model rationalizing synergism between stem cell factor and erythropoietin

Stem cell factor (SCF) and erythropoietin (EPO) are two most recognized growth factors that play in concert to control in vitro erythropoiesis. However, exact mechanisms underlying the interplay of these growth factors in vitro remain unclear. We developed a mathematical model to study co-signaling effects of SCF and EPO utilizing the ERK1/2 and GATA-1 pathways (activated by SCF and EPO) that drive the proliferation and differentiation of erythroid progenitors. The model was simplified and formulated based on three key features: synergistic contribution of SCF and EPO on ERK1/2 activation, positive feedback effects on proliferation and differentiation, and cross-inhibition effects of activated ERK1/2 and GATA-1. The model characteristics were developed to correspond with biological observations made known thus far. Our simulation suggested that activated GATA-1 has a more dominant cross-inhibition effect and stronger positive feedback response on differentiation than the proliferation pathway, while SCF contributed more to the activation of ERK1/2 than EPO. A sensitivity analysis performed to gauge the dynamics of the system was able to identify the most sensitive model parameters and illustrated a contribution of transient activity in EPO ligand to growth factor synergism. Based on theoretical arguments, we have successfully developed a model that can simulate growth factor synergism observed in vitro for erythropoiesis. This hypothesized model can be applied to further computational studies in biological systems where synergistic effects of two ligands are seen.     

Apoptotic cells are generated at every division of in vitro cultured T cell lines.

Long- and short-term T cell lines form the backbone of many assays for T cell function and also represent important tools for use in human immunotherapy. Despite much study concerning the requirements for T cell activation and growth in culture there is relatively little information about the kinetics of proliferation and cell death in such cultures. Here we studied these parameters in a long-term CD8(+) T cell line using a tetrameric MHC reagent and the fluorescent dye CFSE. We observed proliferation of the T cells within 24 h of restimulation with antigen and IL-2 and the cells continued to divide once every 12 h on average. Interestingly, a proportion of cells entered apoptosis with each cell division, showing that a degree of programmed cell death occurred constantly in vitro, not merely at the end of the culture period when antigen or the necessary growth factors became limiting. This information should assist in the design of more efficient protocols for generating large numbers of specific T cells for clinical use.     

Flow cytometric analysis of Lactobacillus plantarum to monitor lag times, cell divisionand injury

Flow cytometry in combination with fluorescent molecular markers 5- (and 6-)carboxyfluorescein succinimidylester (CFSE) and propidium iodide (PI) have been applied todetermine lag times, numbers of cell divisions and injury after mild heat (50°C, 5 min) andnisin treatments (0·1 and 1·0 μg ml⁻¹) of Lactobacillus plantarum. Initial labelling with covalently bound dye CFSE (20 and 100 μg ml⁻¹)allowed determination of lag times and cell proliferation for up to eight generations.Double-labelling with CFSE and PI (5 μg ml⁻¹) provided additional informationabout damage levels and distributions within populations. Subpopulations surviving treatmentcould be identified easily and selectively sorted.     

Immune monitoring in xenotransplantation: the multiparameter flow cytometric mixed lymphocyte culture assay

Xenotransplantation requires monitoring of complex cellular interactions in vitro. A tool to monitor cell proliferation in detail would be instrumental in understanding these cellular interactions in heterogeneous xenogeneic lymphocyte cultures and in patients after xenotransplantation. To accomplish this, we used a fluorescent cell proliferation marker, 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE), in combination with flow cytometry. CFSE, a green fluorescent molecule, binds covalently to intracellular macromolecules. Each cell division reduces the fluorescent intensity per cell by half and shows a characteristic multipeak pattern in flow cytometric analysis. For this study, human lymphocytes were labeled with CFSE and cultured in the presence of irradiated porcine lymphocytes. Cell proliferation was detected in CFSE-labeled lymphocytes in both a single and a multiparameter flow cytometry setting. Concurrently, tritiated ((3)H) thymidine incorporation, a common method to measure gross cell proliferation, was assessed. The kinetics of CFSE-labeled cell proliferation correlated with (3)H-thymidine incorporation in that both methods showed a lag phase for days 1-3 and a log phase for days 4-7. Multiparameter flow cytometric monitoring of mixed lymphocyte cultures allowed phenotyping and assessment of viability of proliferating populations in heterogeneous xenogeneic stimulated human lymphocyte cultures and complemented the classical (3)H-thymidine incorporation assay. The use of this technique will allow a wide array of immunologic parameters to be measured in a heterogeneous xenogeneic mixed lymphocyte culture. The information gained from these assays is essential to understanding the biological significance of xenogeneic cellular interaction and for monitoring the immune status of the xenotransplanted patient.     

Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data

Cellular proliferation is an essential feature of the adaptive immune response. The introduction of the division tracking dye carboxyfluorescein diacetate succinimidyl ester (CFSE) has made it possible to monitor the number of cell divisions during proliferation and to examine the relationship between proliferation and differentiation. Although qualitative examination of CFSE data may be useful, substantially more information about division and death rates can be extracted from quantitative CFSE time-series experiments. Quantitative methods can reveal in detail how lymphocyte proliferation and survival are regulated and altered by signals such as those received from co-stimulatory molecules, drugs and genetic polymorphisms. In this protocol, we present a detailed method for examining time-series data using graphical and computer-based procedures available to all experimenters.     

Magnesium and phosphate enrichment of culture medium stimulates the proliferation of epidermal cells from newborn and adult mice

The proliferation and differentiation of mouse epidermal cells can be sequentially analyzed by modification of extracellular calcium. Newborn cells cultured in low calcium medium (less than 0.1 mM) proliferate as a monolayer and maintain a typical basal cell phenotype in culture but have a limited proliferative capacity and short lifespan. Elevation of the magnesium content of the culture medium from 1 to 5 mM stimulated the proliferation of newborn mouse (1-3 days old) keratinocytes. Maximal DNA synthesis rates, as determined on day 5 of culture, were up to 2-3-fold higher in the magnesium-enriched cultures. Exposure to high magnesium caused 3-4-fold increases in the DNA content of newborn keratinocyte cultures, and extended the confluent phase of epidermal cell growth to over 10 days. Other divalent cations (strontium, copper, zinc, nickel, beryllium, and barium) did not improve keratinocyte growth in culture. Keratinocytes from the tail skin of adult (3 months old) mice displayed an absolute requirement for high phosphate in the culture medium. The medium containing an optimal (10 mM) phosphate concentration prevented the cell detachment caused by the standard low (1 mM) phosphate medium, and in combination with an elevated magnesium content (10-15 mM) it markedly increased both DNA synthesis rates and DNA content of the adult cell cultures. Optimally growing, newborn or adult cultures contained less cells in the G1 phase of the cell cycle and more cells in S and G2 +M. The addition of phosphate and magnesium per se did not induce keratinocyte differentiation and did not interfere with the high calcium (1 mM)-induced differentiation.     

脐血造血细胞的体外扩增：2.造血细胞生长因子和培养液更换的作用

研究了造血干细胞生长因子、白介素-3、白介素-6、粒-巨噬细胞集落刺激因子、粒细胞集落刺激因子及红细胞生成素对脐血造血细胞体外培养的影响及其剂量关系,考察了造血细胞因子单独与联合作用对造血细胞体外培养的影响,证实细胞因子组合使用比细胞因子单独使用效果更好,发现SCF+IL-3+IL-6+GM-CSF+G-CSF+EPO组合对总细胞扩增最佳,SCF+IL-3+IL6+GM-CSF组合对CFU-GM扩增最佳。实验发现培养液更换可大大提高脐血造血细胞总数和祖细胞数产出。在每天更换50%培养液下,脐血总细胞数在第三周扩增了27倍,祖细胞数扩增了21倍。     

Effect of culture pH on erythropoietin production by Chinese hamster ovary cells grown in suspension at 32.5 and 37.0 degrees C

To investigate the effect of culture pH in the range of 6.85-7.80 on cell growth and erythropoietin (EPO) production at 32.5 and 37.0 degrees C, serum-free suspension cultures of recombinant CHO cells (rCHO) were performed in a bioreactor with pH control. Lowering culture temperature from 37.0 to 32.5 degrees C suppressed cell growth, but cell viability remained high for a longer culture period. Regardless of culture temperature, the highest specific growth rate (mu) and maximum viable cell concentration were obtained at pH values of 7.00 and 7.20, respectively. Like mu, the specific consumption rates of glucose and glutamine decreased at 32.5 degrees C compared to 37.0 degrees C. In addition, they increased with increasing culture pH. Culture pH at 32.5 degrees C affected specific EPO productivity (q(EPO)) in a different fashion from that at 37 degrees C. At 37 degrees C, the q(EPO) was fairly constant in the pH range of 6.85-7.80, while at 32.5 degrees C, the q(EPO) was significantly influenced by culture pH. The highest q(EPO) was obtained at pH 7.00 and 32.5 degrees C, and its value was approximately 1.5-fold higher than that at pH 7.00 and 37.0 degrees C. The proportion of acidic EPO isoforms, which is a critical factor for high in vivo biological activity of EPO, was highest in the stationary phase of growth, regardless of culture temperature and pH. Although cell viability rapidly decreased in death phase at both 32.5 and 37.0 degrees C, the significant degradation of produced EPO, probably by the action of proteases released from lysed cells, was observed only at 37.0 degrees C. Taken together, through the optimization of culture temperature and pH, a 3-fold increase in maximum EPO concentration and a 1.4-fold increase in volumetric productivity were obtained at pH 7.00 and 32.5 degrees C when compared with those at 37.0 degrees C. These results demonstrate the importance of optimization of culture temperature and pH for enhancing EPO production in serum-free, suspension culture of rCHO cells.     

Effects of Erythropoietin in Murine-Induced Pluripotent Cell-Derived Panneural Progenitor Cells

Induced cell fate changes by reprogramming of somatic cells offers an efficient strategy to generate autologous pluripotent stem (iPS) cells from any adult cell type. The potential of iPS cells to differentiate into various cell types is well established, however the efficiency to produce functional neurons from iPS cells remains modest. Here, we generated panneural progenitor cells (pNPCs) from mouse iPS cells and investigated the effect of the neurotrophic growth factor erythropoietin (EPO) on their survival, proliferation and neurodifferentiation. Under neural differentiation conditions, iPS-derived pNPCs gave rise to microtubule-associated protein-2 positive neuronlike cells (34% to 43%) and platelet-derived growth factor receptor positive oligodendrocytelike cells (21% to 25%) while less than 1% of the cells expressed the astrocytic marker glial fibrillary acidic protein. Neuronlike cells generated action potentials and developed active presynaptic terminals. The pNPCs expressed EPO receptor (EPOR) mRNA and displayed functional EPOR signaling. In proliferating cultures, EPO (0.1–3 U/mL) slightly improved pNPC survival but reduced cell proliferation and neurosphere formation in a concentration-dependent manner. In differentiating cultures EPO facilitated neurodifferentiation as assessed by the increased number of β-III-tubulin positive neurons. Our results show that EPO inhibits iPS pNPC self-renewal and promotes neurogenesis.