Morphofunctional Properties of Corneal Stromal Cells.

Human corneal stromal cells were isolated by enzymatic digestion from a new source, lenticules obtained during laser vision correction by the ReLEx SMILe method. The resulting culture was mainly presented by fibroblast-like cells with a phenotype CD90-/CD73+/CD105+/keratocan-/lumican-/ALDH1A1+ that differentiate into keratocytes in a specialized medium. The concentration of fetal calf serum-derived growth factors affects the rate of proliferation, production of erythropoietin and brain neurotrophic factor by corneal fibroblasts, and to a lesser extent, their migration activity and production of extracellular matrix components. Thus, the high functional potential of fibroblast-like cells isolated from stromal lenticles can be used to develop cell technologies in ophthalmology.

High Throughput miRNA Screening Identifies miR-574-3p Hyperproductive Effect in CHO Cells.

CHO is the cell line of choice for the manufacturing of many complex biotherapeutics. The constant upgrading of cell productivity is needed to meet the growing demand for these life-saving drugs. Manipulation of small non-coding RNAs-miRNAs-is a good alternative to a single gene knockdown approach due to their post-transcriptional regulation of entire cellular pathways without posing translational burden to the production cell. In this study, we performed a high-throughput screening of 2042-human miRNAs and identified several candidates able to increase cell-specific and overall production of Erythropoietin and Etanercept in CHO cells. Some of these human miRNAs have not been found in Chinese hamster cells and yet were still effective in them. We identified miR-574-3p as being able, when overexpressed in CHO cells, to improve overall productivity of Erythropoietin and Etanercept titers from 1.3 to up to 2-fold. In addition, we validated several targets of miR-574-3p and identified p300 as a main target of miR-574-3p in CHO cells. Furthermore, we demonstrated that stable CHO cell overexpressing miRNAs from endogenous CHO pri-miRNA sequences outperform the cells with human pri-miRNA sequences. Our findings highlight the importance of flanking genomic sequences, and their secondary structure features, on pri-miRNA processing offering a novel, cost-effective and fast strategy as a valuable tool for efficient miRNAs engineering in CHO cells.

Molecular Insights into the Oxygen-Sensing Pathway and Erythropoietin Expression Regulation in Erythropoiesis.

Erythropoiesis is regulated by several factors, including the oxygen-sensing pathway as the main regulator of erythropoietin (EPO) synthesis in the kidney. The release of EPO from the kidney and its binding to the EPO receptor (EPOR) on erythrocyte progenitor cells in the bone marrow results in increased erythropoiesis. Any imbalance in these homeostatic mechanisms can lead to dysregulated erythropoiesis and hematological disorders. For example, mutations in genes encoding key players of oxygen-sensing pathway and regulation of EPO production (HIF-EPO pathway), namely VHL, EGLN, EPAS1 and EPO, are well known causative factors that contribute to the development of erythrocytosis. We aimed to investigate additional molecular mechanisms involved in the HIF-EPO pathway that correlate with erythropoiesis. To this end, we conducted an extensive literature search and used several in silico tools. We identified genes encoding transcription factors and proteins that control transcriptional activation or repression; genes encoding kinases, deacetylases, methyltransferases, conjugating enzymes, protein ligases, and proteases involved in post-translational modifications; and genes encoding nuclear transport receptors that regulate nuclear transport. All these genes may modulate the stability or activity of HIF2α and its partners in the HIF-EPO pathway, thus affecting EPO synthesis. The theoretical information we provide in this work can be a valuable tool for a better understanding of one of the most important regulatory pathways in the process of erythropoiesis. This knowledge is necessary to discover the causative factors that may contribute to the development of hematological diseases and improve current diagnostic and treatment solutions in this regard.

Regulated control of gene therapies by drug-induced splicing.

So far, gene therapies have relied on complex constructs that cannot be finely controlled1,2. Here we report a universal switch element that enables precise control of gene replacement or gene editing after exposure to a small molecule. The small-molecule inducers are currently in human use, are orally bioavailable when given to animals or humans and can reach both peripheral tissues and the brain. Moreover, the switch system, which we denote Xon, does not require the co-expression of any regulatory proteins. Using Xon, the translation of the desired elements for controlled gene replacement or gene editing machinery occurs after a single oral dose of the inducer, and the robustness of expression can be controlled by the drug dose, protein stability and redosing. The ability of Xon to provide temporal control of protein expression can be adapted for cell-biology applications and animal studies. Additionally, owing to the oral bioavailability and safety of the drugs used, the Xon switch system provides an unprecedented opportunity to refine and tailor the application of gene therapies in humans.

Effect of trivalent chromium on erythropoietin production and the prevention of insulin resistance in HepG2 cells.

In erythropoietin (EPO)-producing HepG2 cells, we investigated the effect of trivalent chromium (Cr) on the promotion of EPO production and the induction of insulin resistance. Cr increased hypoxia-inducible factor (HIF)-1α protein, EPO mRNA expression and EPO protein levels in HepG2 cells. The effect of Cr on EPO production was inhibited by inhibition of proliferator-activated receptor γ (PPARγ). Insulin resistance was induced by culturing with insulin resistance induction medium supplemented with palmitic acid for 24 h. When Cr was added to the medium, the increase in glucose-6-phosphatase and phosphoenolpyruvate carboxykinase 1 mRNA expression levels and the decrease in the ratio of phosphorylated Akt to Akt protein were suppressed, and the induction of insulin resistance prevented. When a PPARγ inhibitor or siPPARγ was added together with Cr, the inhibitory effect of Cr on the induction of insulin resistance disappeared. In addition, pretreatment with siEPO suppressed the increase in EPO mRNA expression, and the inhibitory effect on the induction of insulin resistance due to the addition of Cr was significantly reduced. These results suggest that the inhibition of insulin resistance induction by Cr in HepG2 cells involves the promotion of EPO production mediated by PPARγ, in addition to other PPARγ-mediated activities.

Roxadustat for dialysis patients with erythropoietin hypo-responsiveness: a single-center, prospective investigation.

Dialysis patients with erythropoietin hypo-responsiveness suffered from refractory anemia. Roxadustat reversibly binds and inhibits hypoxia-inducible factor-prolyl hydroxylase (HIF-PHD), resulting in increased endogenous EPO which stimulates erythropoiesis, theoretically has an advantage over exogenous EPO in anti-anemia therapy. From September 2019 to October 2020, 32 dialysis patients with hypo-responsiveness to erythropoietin were evaluated. During the 24-week follow-up period, all patients were taken off erythropoietin and switched to roxadustat. Dosage adjustments were administrated according to the fluctuation of hemoglobin level during the treatment. Parameters about anemia, iron metabolism and biochemical indexes were collected, and adverse events were recorded. A total of 31 patients completed the clinical observation, with varying degrees of malnutrition-inflammation. Post treatment, the levels of transferrin and total iron-binding capacity were increased, while that of transferrin saturation and cholesterol decreased. 15 cases (accounting for 48.39%, designated as fulfilled group) met the target level of hemoglobin, while 16 cases (51.61%, non-fulfilled group) did not. The baseline conditions of the above two groups were compared. The levels of hypersensitive C-reactive protein, interleukin-6 and serum ferritin in the non-fulfilled group were higher than those in the fulfilled group, and the levels of residual renal function, serum albumin, iron, transferrin and total iron-binding capacity were lower than those in the fulfilled group. Linear regression analysis showed that increase of HsCRP had a negative effect on the improvement of Hb. One case of adverse reaction grade 3 and four cases of grade 2 occurred throughout the study, yet all were relieved after therapy. Significant anti-anemia effects could be achieved in most patients with erythropoietin hypo-responsiveness after treatment with roxadustat, accompanied by relatively mild and rare adverse reactions. The malnutrition-inflammation states of patients may interfere with the anti-anemia effect of roxadustat, and iron utilization is more important than iron storage in anemia improvement.

Retinoic acid regulates erythropoietin production cooperatively with hypoxia-inducible factors in human iPSC-derived erythropoietin-producing cells.

Erythropoietin (EPO) is a crucial hormone for erythropoiesis and produced by adult kidneys. Insufficient EPO production in chronic kidney disease (CKD) can cause renal anemia. Although hypoxia-inducible factors (HIFs) are known as a main regulator, the mechanisms of EPO production have not been fully elucidated. In this study, we aimed to examine the roles of retinoic acid (RA) in EPO production using EPO-producing cells derived from human induced pluripotent stem cells (hiPSC-EPO cells) that we previously established. RA augmented EPO production by hiPSC-EPO cells under hypoxia or by treatment with prolyl hydroxylase domain-containing protein (PHD) inhibitors that upregulate HIF signals. Combination treatment with RA and a PHD inhibitor improved renal anemia in vitamin A-depleted CKD model mice. Our findings using hiPSC-EPO cells and CKD model mice may contribute to clarifying the EPO production mechanism and developing efficient therapies for renal anemia.

CD34-Positive Blast Count and p53 Expression in Bone Marrow Biopsies of Patients with Low-Risk Myelodysplastic Syndromes: Potential Predictive Tools of Response to Erythropoietin Stimulating Agents.

INTRODUCTION: The first-line therapy for patients with low-risk myelodysplastic syndromes (MDSs) commonly consists of erythropoietin stimulating agents (ESAs), with a response rate ranging from 34 to 62%. For nonresponder patients, outside clinical trials, blood transfusions are the most frequent therapeutic option, with detrimental effect on the quality of life and with risks of iron-overload. Since no studies have been yet conducted on this topic, we investigated the potential predictive role of bone marrow (BM) histological evaluation in patients treated with ESAs. MATERIALS AND METHODS: We performed a morphological and immunohistochemical retrospective analysis of BM biopsies of 96 patients with low-risk MDSs subsequently treated with ESAs. RESULTS: In our series, substantial morphological overlap was found between responder and nonresponder patients. On the contrary, patients with a percentage of CD34-positive blasts >3% or with p53 protein expression <1% responded with a significantly higher frequency to ESAs. CONCLUSIONS: Our study reinforces the role of BM biopsy as diagnostic tool in MDSs, being also able to supply information related to response to ESAs and to its loss over time.

Renal interstitial fibroblasts coproduce erythropoietin and renin under anaemic conditions.

BACKGROUND: Erythrocyte mass contributes to maintaining systemic oxygen delivery and blood viscosity, with the latter being one of the determinants of blood pressure. However, the physiological response to blood pressure changes under anaemic conditions remain unknown. METHODS AND FINDINGS: We show that anaemia decreases blood pressure in human patients and mouse models. Analyses of pathways related to blood pressure regulation demonstrate that anaemia enhances the expression of the gene encoding the vasopressor substance renin in kidneys. Although kidney juxtaglomerular cells are known to continuously produce renin, renal interstitial fibroblasts are identified in the present study as a novel site of renin induction under anaemic hypotensive conditions in mice and rats. Notably, some renal interstitial fibroblasts are found to simultaneously express renin and the erythroid growth factor erythropoietin in the anaemic mouse kidney. Antihypertensive agents but not hypoxic stimuli induced interstitial renin expression, suggesting that blood pressure reduction triggers interstitial renin induction in anaemic mice. The interstitial renin expression was also detected in injured fibrotic kidneys of the mouse and human, and the renin-expressing interstitial cells in murine fibrotic kidneys were identified as myofibroblasts originating from renal interstitial fibroblasts. Since the elevated expression levels of renin in fibrotic kidneys along with progression of renal fibrosis were well correlated to the systemic blood pressure increase, the renal interstitial renin production seemed to affect systemic blood pressure. INTERPRETATION: Renal interstitial fibroblasts function as central controllers of systemic oxygen delivery by producing both renin and erythropoietin. FUNDING: Grants-in-Aid from Japan Society for the Promotion of Science (JSPS) KAKENHI (17K19680, 15H04691, and 26111002) and the Takeda Science Foundation.

Development of Magoh protein-overexpressing HEK cells for optimized therapeutic protein production.

In the pharmaceutical industry, the need for high levels of protein expression in mammalian cells has prompted the search for new strategies, including technologies to obtain cells with improved mechanisms that enhance its transcriptional activity, folding, or protein secretion. Chinese Hamster Ovary (CHO) cells are by far the most used host cell for therapeutic protein expression. However, these cells produce specific glycans that are not present in human cells and therefore potentially immunogenic. As a result, there is an increased interest in the use of human-derived cells for therapeutic protein production. For many decades, human embryonic kidney (HEK) cells were exclusively used for research. However, two products for therapeutic indication were recently approved in the United States. It was previously shown that tethered Magoh, an Exon-junction complex core component, to specific mRNA sequences, have had significant positive effects on mRNA translational efficiency. In this study, a HEK Magoh-overexpressing cell line and clones, designated here as HEK-MAGO, were developed for the first time. These cells exhibited improved characteristics in protein expression, reaching -two- to threefold increases in rhEPO protein production in comparison with the wild-type cells. Moreover, this effect was promoter independent highlighting the versatility of this expression platform.

G-CSF shifts erythropoiesis from bone marrow into spleen in the setting of systemic inflammation.

The anemia of inflammation is related in part to abnormal erythropoiesis in bone marrow. G-CSF regulates granulopoiesis and is increased during systemic inflammation. Here, we have showed that high levels of G-CSF are associated with repression of bone marrow erythropoiesis and expansion of splenic erythropoiesis in Escherichia coli-infected mice and lipopolysaccharide-treated mice. Under lipopolysaccharide-induced systemic inflammatory conditions in mice, G-CSF neutralization with antibody alleviated the blockage of bone marrow erythropoiesis, prevented the enhancement of splenic erythropoiesis, ameliorated splenomegaly, and reduced the brittleness of spleen. We further demonstrated that after lipopolysaccharide treatment, TLR4-knockout mice display low levels of G-CSF, healthy bone marrow erythropoiesis, almost no stress erythropoiesis in the spleen, and normal size and toughness of spleen. In addition, we found HIF-mediated erythropoietin production is essential for splenic erythropoiesis in the setting of G-CSF-induced suppression of bone marrow erythropoiesis. Our findings identify G-CSF as a critical mediator of inflammation-associated erythropoiesis dysfunction in bone marrow and offer insight into the mechanism of G-CSF-induced splenic erythropoiesis. We provide experimentally significant dimension to the biology of G-CSF.

Erythropoietin regulates metabolic response in mice via receptor expression in adipose tissue, brain, and bone.

Erythropoietin (EPO) acts by binding to erythroid progenitor cells to regulate red blood cell production. While EPO receptor (Epor) expression is highest on erythroid tissue, animal models exhibit EPO activity in nonhematopoietic tissues, mediated, in part, by tissue-specific Epor expression. This review describes the metabolic response in mice to endogenous EPO and EPO treatment associated with glucose metabolism, fat mass accumulation, and inflammation in white adipose tissue and brain during diet-induced obesity and with bone marrow fat and bone remodeling. During high-fat diet-induced obesity, EPO treatment improves glucose tolerance, decreases fat mass accumulation, and shifts white adipose tissue from a pro-inflammatory to an anti-inflammatory state. Fat mass regulation by EPO is sex dimorphic, apparent in males and abrogated by estrogen in females. Cerebral EPO also regulates fat mass and hypothalamus inflammation associated with diet-induced obesity in males and ovariectomized female mice. In bone, EPO contributes to the balance between adipogenesis and osteogenesis in both male and female mice. EPO treatment promotes bone loss mediated via Epor in osteoblasts and reduces bone marrow adipocytes before and independent of change in white adipose tissue fat mass. EPO regulation of bone loss and fat mass is independent of EPO-stimulated erythropoiesis. EPO nonhematopoietic tissue response may relate to the long-term consequences of EPO treatment of anemia in chronic kidney disease and to the alternative treatment of oral hypoxia-inducible factor prolyl hydroxylase inhibitors that increase endogenous EPO production.

Erythropoietin production by the kidney and the liver in response to severe hypoxia evaluated by Western blotting with deglycosylation.

The detection of erythropoietin (Epo) protein by Western blotting has required pre-purification of the sample. We developed a new Western blot method to detect plasma and urinary Epo using deglycosylation. Epo in urine and tissue, and erythropoiesis-stimulating agents (ESAs) in urine were directly detected by our Western blotting. Plasma Epo and ESAs were not detected by direct application but were detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except for PEG-bound epoetin ß pegol. The 22 kDa band from an anemic patient's urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo. Severe hypoxia (7% O2, 4 hr) caused a 400-fold increase in deglycosylated Epo expression in rat kidneys, which is consistent with the increases in both Epo gene expression and plasma Epo concentration. Immunohistochemistry showed Epo expression in nephrons but not in interstitial cells under control conditions, and hypoxia increased Epo expression in interstitial cells but not in tubules. These data show that intrinsic Epo and all ESAs can be detected by Western blot either directly in urine or after deglycosylation in blood, and that the kidney but not the liver is the main site of Epo production in control and severe hypoxia. Our method will make the tests for Epo doping and detection easy.

Prolonged astrocyte-derived erythropoietin expression attenuates neuronal damage under hypothermic conditions.

BACKGROUND: Hypoxic-ischemic encephalopathy (HIE) has a high morbidity rate and involves severe neurologic deficits, including cerebral palsy. Therapeutic hypothermia (TH) has been shown to decrease the mortality rate and provide neuroprotection in infants with HIE. However, death and disability rates in HIE infants treated with TH remain high. Although the cellular mechanism of the neuroprotective effect of TH remains unclear, astrocytic erythropoietin (EPO) is known to be a key mediator of neuroprotection under hypoxic conditions. In the present study, we investigated the hypothermia effect on EPO expression in astrocytes and determined whether hypothermia attenuates neuronal damage via EPO signaling. METHODS: Astrocytes derived from rat cerebral cortex were cultured under oxygen/glucose deprivation (OGD). The expression of EPO and hypoxia-inducible factor (HIF), a transcription factor of EPO, was assessed. After OGD, astrocytes were cultured under normothermic (37 °C) or hypothermic (33.5 °C) conditions, and then EPO and HIF expression was assessed. After OGD, rat cortical neurons were cultured in astrocyte-conditioned medium (ACM) derived from the hypothermic group, and neuronal apoptosis was evaluated. RESULTS: OGD induced EPO mRNA and protein expression, although at lower levels than hypoxia alone. HIF-1α and HIF-2α protein expression increased under hypoxia alone and OGD, although OGD increased HIF-2α protein expression less than hypoxia alone. EPO gene and protein expression after OGD was significantly higher under hypothermia. Moreover, expression of HIF-1α and HIF-2α protein was enhanced under hypothermia. In the presence of ACM derived from hypothermic astrocytes following OGD, the number of cleaved caspase 3 and TdT-mediated dUTP nick-end labeling-positive apoptotic neurons was lower than in the presence of ACM from normothermic astrocytes following OGD. Blockade of EPO signaling using anti-EPO neutralization antibody attenuated the anti-apoptotic effect of ACM derived from hypothermic astrocytes following OGD. CONCLUSIONS: Hypothermia after OGD stabilized HIF-EPO signaling in astrocytes, and upregulated EPO expression could suppress neuronal apoptosis. Investigating the neuroprotective effect of EPO from astrocytes under hypothermic conditions may contribute to the development of novel neuroprotection-based therapies for HIE.

Assessment and streamlined preparation of low-cytotoxicity lentiviral vectors for mobilized human hematopoietic stem cell transduction.

As important vectors for ectopic protein expression, gene silencing, and progenitor cell barcoding, lentiviruses continue to emerge as versatile research and clinical tools. For studies employing cell types that are relatively resistant to transduction, high-titer lentivirus preparations with low cytotoxicity are required. During lentivirus production, carryover plasmid DNA endotoxins, transfection reagents, damaged packaging cells, and virus concentration procedures are potential sources of cytotoxicity. As an often unevaluated property of lentivirus preparations, cytotoxicity can unwittingly skew estimates of functional titers and complicate interpretations of transduced cell phenotypes. By employing hematopoietic UT7epo cells cultured in erythropoietin (EPO) below maximal dosing, we first define a sensitive flow cytometric bioassay for critically assessing the cytotoxicity (and titers) of lentivirus preparations. Bioassay of custom preparations of research-grade lentiviruses from six commercial sources unexpectedly revealed substantial cytotoxicity (with certain preparations additionally registering titers several log below designated values). To overcome such limiting properties, we further report on unique, efficient workflows for reproducibly preparing and processing high-titer, low-cytotoxicity (HTLC) lentiviruses at research scale. These HTLC lentiviruses reliably transduce peripheral blood hematopoietic stem/progenitor cells (PB-HSPCs) at frequencies ≥40%, with low cytotoxicity. In addition, by employing cyclosporin H (to inhibit IFITM3), PB-HSPCs can be transduced at heightened efficiency with nominal cytotoxicity. Overall, this work provides straightforward approaches to (1) critical assessment of the cytotoxicity of lentivirus preparations; (2) reproducible generation (and concentration) of high-quality lentiviruses via a streamlined workflow; and (3) transduction of PB-HSPCs at benchmark levels with nominal cytotoxicity.

HIF­1α attenuates neuronal apoptosis by upregulating EPO expression following cerebral ischemia­reperfusion injury in a rat MCAO model.

Hypoxia­inducible factor­1α (HIF­1α) is a key transcriptional factor in response to hypoxia and is involved in ischemic stroke. In the present study, the potential for HIF­1α to inhibit neuronal apoptosis through upregulating erythropoietin (EPO) was investigated in a transient middle cerebral artery occlusion (tMCAO) rat stroke model. For this purpose, a recombinant adenovirus expressing HIF­1α was engineered (Ad­HIF­1α). Control adenovirus (Ad group), Ad­HIF­1α (Ad­HIF­1α group) or Ad­HIF­1α in addition to erythropoietin mimetic peptide­9 (EMP9), an EPO­receptor (­R) antagonist (Ad­HIF­1α+EMP9 group), were used for an intracranial injection into rat ischemic penumbra 1 h following MCAO. All rats demonstrated functional improvement following tMCAO, while the improvement rate was faster in rats treated by Ad­HIF­1α compared with all other groups. The EPO­R inhibitor partially reversed the benefits of Ad­HIF­1α. Apoptosis induced by tMCAO was significantly inhibited by Ad­HIF­1α (P<0.05). The expression of HIF­1α, evaluated by immunohistochemistry either in neurons or astrocytes, was upregulated by Ad­HIF­1α. Both EPO mRNA and protein expression were increased by Ad­HIF­1α, however, there was no significant change of EPO­R either on an mRNA level or protein level. Furthermore, EMP9 did not change the EPO expression which was upregulated by Ad­HIF­1α. Activated caspase 3 in neurons was suppressed by Ad­HIF­1α. Activated caspase 3 downregulated by HIF­1α was partially blocked by EMP9. Altogether, the present data demonstrated that HIF­1α attenuates neuronal apoptosis partially through upregulating EPO following cerebral ischemia in rat. Thus, upregulating HIF­1α subsequent to a stroke may be a potential treatment for ischemic stroke.

Enhancing the yield of human erythropoietin in Aspergillus niger by introns and CRISPR-Cas9.

Aspergillus niger has been employed to produce heterologous proteins due to its high capacity for expression and secretion; nevertheless, expression levels of human proteins have been modest. We were interested in investigating whether A. niger can express and secret human erythropoietin (HuEPO) at high yields. Our strategy was to combine the presence of introns with CRISPR-Cas9 to increase the yield of the recombinant protein. The epo gene was codon-optimized and its expression driven by the PmbfA promoter. Another version of epo contained introns from the fructose-1,6-bisphosphatase (fbp) gene. Two recombinant clones, uME12 (no introns) and uME23 (with introns), were selected based on the resistance to the antibiotic and because they showed a protein profile different from that of the parental strain, as shown by SDS-PAGE. Expression of epo was confirmed by RT-PCR in both colonies but the recombinant EPO protein (rHUEPO) was detected by Western blot only in uME23. The rHuEPO yield from uME23 was estimated at about 1.8 mg L-1 by ELISA, demonstrating that the presence of introns resulted in higher yield, possibly by conferring more stability to mRNA. On the other hand, as part of our strategy we decided to inactivate in the strain uME23 the following genes vps, prtT, algC and och1 which are involved in protein secretion, regulating of protease expression and protein glycosylation in A. niger, with CRISPR-Cas9, yielding the muPS20 transformant. muPS20 is a protease-free strain and its rHuEPO production level was increased 41.1-fold. Moreover, its molecular weight was ≈27 kDa showing that mutations in the above mentioned genes improved secretion, prevented proteolytic degradation and hyperglycosylation of heterologous protein.

Small-scale bioreactor supports high density HEK293 cell perfusion culture for the production of recombinant Erythropoietin.

Process intensification in mammalian cell culture-based recombinant protein production has been achieved by high cell density perfusion exceeding 108 cells/mL in the recent years. As the majority of therapeutic proteins are produced in Chinese Hamster Ovary (CHO) cells, intensified perfusion processes have been mainly developed for this type of host cell line. However, the use of CHO cells can result in non-human posttranslational modifications of the protein of interest, which may be disadvantageous compared with human cell lines. In this study, we developed a high cell density perfusion process of Human Embryonic Kidney (HEK293) cells producing recombinant human Erythropoietin (rhEPO). Firstly, a small-scale perfusion system from commercial bench-top screening bioreactors was developed for <250 mL working volume. Then, after the first trial runs with CHO cells, the system was modified for HEK293 cells (more sensitive than CHO cells) to achieve a higher oxygen transfer under mild aeration and agitation conditions. Steady states for medium (20 × 106 cells/mL) and high cell densities (80 × 106 cells/mL), normal process temperature (37 °C) and mild hypothermia (33 °C) as well as different cell specific perfusion rates (CSPR) from 10 to 60 pL/cell/day were applied to study the performance of the culture. The volumetric productivity was maximized for the high cell density steady state but decreased when an extremely low CSPR of 10 pL/cell/day was applied. The shift from high to low CSPR strongly reduced the nutrient uptake rates. The results from our study show that human cell lines, such as HEK293 can be used for intensified perfusion processes.

Effects of sorbitol and lactate on erythropoietin production in HepG2 cells.

Promotion of erythropoietin (EPO) production is important for erythropoiesis as well as cell viability. The most effective inducing factor for EPO production is hypoxia. Hypoxia inducible factor (HIF), a regulator of EPO production, is increased under hypoxic conditions and is also affected by various regulators such as sirtuin1 (SIRT1). SIRT1 is regulated by the cytoplasmic redox state, which is thought to affect EPO production. Therefore, we investigated the effects of sorbitol and lactic acid, which serve as substrates for cellular respiration and bring cells into a reduced state, on EPO production in HepG2 cells. The addition of low-concentration sorbitol to HepG2 cells produced a mildly reduced state similar to that of hypoxia and increased NAD+, SIRT1, and HIF-α, and EPO mRNA expression. On the other hand, lactate suppressed EPO mRNA expression at all concentrations. Inhibition of lactate production from pyruvate abolished the effect of low sorbitol concentrations on EPO mRNA expression. When low-concentration sorbitol and a reducing agent were administered simultaneously, the effect of increasing EPO mRNA expression disappeared. It was suggested that SIRT1 and EPO production increased under conditions where lactate production was not suppressed, even under mildly reduced conditions similar to hypoxia.