Circulating tumor extracellular vesicles to monitor metastatic prostate cancer genomics and transcriptomic evolution.

Extracellular vesicles (EVs) secreted by tumors are abundant in plasma, but their potential for interrogating the molecular features of tumors through multi-omic profiling remains widely unexplored. Genomic and transcriptomic profiling of circulating EV-DNA and EV-RNA isolated from in vitro and in vivo models of metastatic prostate cancer (mPC) reveal a high contribution of tumor material to EV-loaded DNA/RNA, validating the findings in two cohorts of longitudinal plasma samples collected from patients during androgen receptor signaling inhibitor (ARSI) or taxane-based therapy. EV-DNA genomic features recapitulate matched-patient biopsies and circulating tumor DNA (ctDNA) and associate with clinical progression. We develop a novel approach to enable transcriptomic profiling of EV-RNA (RExCuE). We report how the transcriptome of circulating EVs is enriched for tumor-associated transcripts, captures certain patient and tumor features, and reflects on-therapy tumor adaptation changes. Altogether, we show that EV profiling enables longitudinal transcriptomic and genomic profiling of mPC in liquid biopsy.

Late poor outcomes of Clostridioides difficile infections in oncological patients: A multicentre cohort study.

Background: Cancer has been associated with an increased risk of in-hospital mortality in CDI patients. However, data on delayed mortality in cancer patients with CDI are scarce. Aim/Objective: The aim of the present study was to compare outcomes between oncological patients and the general population with Clostridioides difficile infection (CDI) after 90 days of follow-up. Methods: A multicenter prospective cohort study was conducted in 28 hospitals participating in the VINCat program. Cases were all consecutive adult patients who met the case definition of CDI. Sociodemographic, clinical, and epidemiological variables and evolution at discharge and after 90 days were recorded for each case. Findings/results: The mortality rate was higher in oncological patients (OR = 1.70, 95% CI: 1.08-2.67). In addition, oncological patients receiving chemotherapy (CT) presented higher recurrence rates (18.5% vs 9.8%, p = 0.049). Among oncological patients treated with metronidazole, those with active CT showed a higher rate of recurrence (35.3% vs 8.0% p = 0.04). Discussion: Oncological patients presented a higher risk of poor outcomes after CDI. Their early and late mortality rates were higher than in the general population, and in parallel, those undergoing chemotherapy (especially those receiving metronidazole) had higher rates of recurrence.

Development of Antifouling Polysulfone Membranes by Synergistic Modification with Two Different Additives in Casting Solution and Coagulation Bath: Synperonic F108 and Polyacrylic Acid.

This study deals with the development of antifouling ultrafiltration membranes based on polysulfone (PSF) for wastewater treatment and the concentration and purification of hemicellulose and lignin in the pulp and paper industry. The efficient simple and reproducible technique of PSF membrane modification to increase antifouling performance by simultaneous addition of triblock copolymer polyethylene glycol-polypropylene glycol-polyethylene glycol (Synperonic F108, Mn =14 × 103 g mol-1) to the casting solution and addition of polyacrylic acid (PAA, Mn = 250 × 103 g mol-1) to the coagulation bath is proposed for the first time. The effect of the PAA concentration in the aqueous solution on the PSF/Synperonic F108 membrane structure, surface characteristics, performance, and antifouling stability was investigated. PAA concentrations were varied from 0.35 to 2.0 wt.%. Membrane composition, structure, and topology were investigated by Fourier-transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and scanning electron microscopy (SEM). The addition of PAA into the coagulation bath was revealed to cause the formation of a thicker and denser selective layer with decreasing its pore size and porosity; according to the structural characterization, an interpolymer complex of the two additives was formed on the surface of the PSF membrane. Hydrophilicity of the membrane selective layer surface was shown to increase significantly. The selective layer surface charge was found to become more negative in comparison to the reference membrane. It was shown that PSF/Synperonic F108/PAA membranes are characterized by better antifouling performance in ultrafiltration of humic acid solution and thermomechanical pulp mill (ThMP) process water. Membrane modification with PAA results in higher ThMP process water flux, fouling recovery ratio, and hemicellulose and total lignin rejection compared to the reference PSF/Synperonic F108 membrane. This suggests the possibility of applying the developed membranes for hemicellulose concentration and purification.

Extracellular Vesicle and Particle Biomarkers Define Multiple Human Cancers.

There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.

Tumour exosomal CEMIP protein promotes cancer cell colonization in brain metastasis.

The development of effective therapies against brain metastasis is currently hindered by limitations in our understanding of the molecular mechanisms driving it. Here we define the contributions of tumour-secreted exosomes to brain metastatic colonization and demonstrate that pre-conditioning the brain microenvironment with exosomes from brain metastatic cells enhances cancer cell outgrowth. Proteomic analysis identified cell migration-inducing and hyaluronan-binding protein (CEMIP) as elevated in exosomes from brain metastatic but not lung or bone metastatic cells. CEMIP depletion in tumour cells impaired brain metastasis, disrupting invasion and tumour cell association with the brain vasculature, phenotypes rescued by pre-conditioning the brain microenvironment with CEMIP+ exosomes. Moreover, uptake of CEMIP+ exosomes by brain endothelial and microglial cells induced endothelial cell branching and inflammation in the perivascular niche by upregulating the pro-inflammatory cytokines encoded by Ptgs2, Tnf and Ccl/Cxcl, known to promote brain vascular remodelling and metastasis. CEMIP was elevated in tumour tissues and exosomes from patients with brain metastasis and predicted brain metastasis progression and patient survival. Collectively, our findings suggest that targeting exosomal CEMIP could constitute a future avenue for the prevention and treatment of brain metastasis.

Successful Treatment of Endometriosis-Related Hemorrhagic Ascites: A Report of Three Cases.

Endometriosis-related ascites is rare and is frequently confused with an ovarian malignancy. Since it affects women in reproductive age, its diagnosis and therapy are even more challenging. These patients usually present with abdominal distension, pelvic pain, and weight loss, but a careful questioning usually reveals the typical endometriosis symptoms-such as dysmenorrhea and dyspareunia. We present three cases of endometriosis-related ascites, one of them with pleural effusion. All cases were associated with extensive disease and required laborious laparoscopic surgery, medical therapy with gonadotropin releasing hormone analogs, and long-term follow-up. One of the patients delivered twins following an in vitro fertilization (IVF) cycle without recurrence of ascites. We aim to raise awareness toward the importance of considering endometriosis in a patient with ascites of unknown origin.

Hydrogen peroxide regulates angiogenesis-related factors in tumor cells.

Tumor angiogenesis is required for tumor development and growth, and is regulated by several factors including ROS. H2O2 is a ROS with an important role in cell signaling, but how H2O2 regulates tumor angiogenesis is still poorly understood. We have xenografted tumor cells with altered levels of H2O2 by catalase overexpression into zebrafish embryos to study redox-induced tumor neovascularization. We found that vascular recruitment and invasion were impaired if catalase was overexpressed. In addition, the overexpression of catalase altered the transcriptional levels of several angiogenesis-related factors in tumor cells, including TIMP-3 and THBS1. These two anti-angiogenic factors were found to be H2O2-regulated by two different mechanisms: TIMP-3 expression in a cell-autonomous manner; and, THBS1 expression that was non-cell-autonomous. Our work shows that intracellular H2O2 regulates the expression of angiogenic factors and the formation of a vessel network. Understanding the molecular mechanisms that govern this multifunctional effect of H2O2 on tumor angiogenesis could be important for the development of more efficient anti-angiogenic therapies.

Engineered hydrogels increase the post-transplantation survival of encapsulated hESC-derived midbrain dopaminergic neurons.

Cell replacement therapies have broad biomedical potential; however, low cell survival and poor functional integration post-transplantation are major hurdles that hamper clinical benefit. For example, following striatal transplantation of midbrain dopaminergic (mDA) neurons for the treatment of Parkinson's disease (PD), only 1-5% of the neurons typically survive in preclinical models and in clinical trials. In general, resource-intensive generation and implantation of larger numbers of cells are used to compensate for the low post-transplantation cell-survival. Poor graft survival is often attributed to adverse biochemical, mechanical, and/or immunological stress that cells experience during and after implantation. To address these challenges, we developed a functionalized hyaluronic acid (HA)-based hydrogel for in vitro maturation and central nervous system (CNS) transplantation of human pluripotent stem cell (hPSC)-derived neural progenitors. Specifically, we functionalized the HA hydrogel with RGD and heparin (hep) via click-chemistry and tailored its stiffness to encourage neuronal maturation, survival, and long-term maintenance of the desired mDA phenotype. Importantly, ∼5 times more hydrogel-encapsulated mDA neurons survived after transplantation in the rat striatum, compared to unencapsulated neurons harvested from commonly used 2D surfaces. This engineered biomaterial may therefore increase the therapeutic potential and reduce the manufacturing burden for successful neuronal implantation.

Defined and Scalable Differentiation of Human Oligodendrocyte Precursors from Pluripotent Stem Cells in a 3D Culture System.

Oligodendrocyte precursor cells (OPCs) offer considerable potential for the treatment of demyelinating diseases and injuries of the CNS. However, generating large quantities of high-quality OPCs remains a substantial challenge that impedes their therapeutic application. Here, we show that OPCs can be generated from human pluripotent stem cells (hPSCs) in a three-dimensional (3D), scalable, and fully defined thermoresponsive biomaterial system. We used CRISPR/Cas9 to create a NKX2.2-EGFP human embryonic stem cell reporter line that enabled fine-tuning of early OPC specification and identification of conditions that markedly increased the number of OLIG2+ and NKX2.2+ cells generated from hPSCs. Transplantation of 50-day-old OPCs into the brains of NOD/SCID mice revealed that progenitors generated in 3D without cell selection or purification subsequently engrafted, migrated, and matured into myelinating oligodendrocytes in vivo. These results demonstrate the potential of harnessing lineage reporter lines to develop 3D platforms for rapid and large-scale production of OPCs.

Pre-metastatic niches: organ-specific homes for metastases.

It is well established that organs of future metastasis are not passive receivers of circulating tumour cells, but are instead selectively and actively modified by the primary tumour before metastatic spread has even occurred. Sowing the 'seeds' of metastasis requires the action of tumour-secreted factors and tumour-shed extracellular vesicles that enable the 'soil' at distant metastatic sites to encourage the outgrowth of incoming cancer cells. In this Review, we summarize the main processes and new mechanisms involved in the formation of the pre-metastatic niche.

Targeted gene knock-in by homology-directed genome editing using Cas9 ribonucleoprotein and AAV donor delivery.

Realizing the full potential of genome editing requires the development of efficient and broadly applicable methods for delivering programmable nucleases and donor templates for homology-directed repair (HDR). The RNA-guided Cas9 endonuclease can be introduced into cells as a purified protein in complex with a single guide RNA (sgRNA). Such ribonucleoproteins (RNPs) can facilitate the high-fidelity introduction of single-base substitutions via HDR following co-delivery with a single-stranded DNA oligonucleotide. However, combining RNPs with transgene-containing donor templates for targeted gene addition has proven challenging, which in turn has limited the capabilities of the RNP-mediated genome editing toolbox. Here, we demonstrate that combining RNP delivery with naturally recombinogenic adeno-associated virus (AAV) donor vectors enables site-specific gene insertion by homology-directed genome editing. Compared to conventional plasmid-based expression vectors and donor templates, we show that combining RNP and AAV donor delivery increases the efficiency of gene addition by up to 12-fold, enabling the creation of lineage reporters that can be used to track the conversion of striatal neurons from human fibroblasts in real time. These results thus illustrate the potential for unifying nuclease protein delivery with AAV donor vectors for homology-directed genome editing.

Influence of interhospital transfer on outcomes of symptomatic and ruptured abdominal aortic aneurysms.

Objetive: Evaluate the influence of the geographic location of patients with symptomatic abdominal aortic aneurysms (AAA) or ruptured AAA (rAAA), on mortality. METHODS: Retrospective review of all cases of symptomatic AAA and rAAA submitted to surgery in a tertiary institution, between January 2011 and August 2017. The main outcome was in-hospital mortality. Secondary outcomes were admission to intensive care unit (ICU), length of ICU and hospital stay, type of repair and anesthesia and weekend presentation. Data was submitted to univariable analysis and logistic regression. Statistical significance was considered if the p value was <0.05. RESULTS: 135 patients were admitted with the diagnosis of symptomatic or rAAA and submitted to surgery, 83 (61.5%) by endovascular repair and 52 (38.5%) by open repair, 30.4% with local anesthesia and sedation. 92 patients (68.1%) were transferred from other hospitals, with a mean distance of 113±88 km. Subgroup analysis revealed that there were no significant differences between transferred and not transferred patients' groups concerning main outcome (31.5% vs 34.9%, p=0.35), baseline characteristics (age and gender), type of surgery and anesthesia, weekend presentation, ICU admission, length of ICU and hospital stay. Logistic regression analysis revealed that the variables associated with mortality were female gender (odds ratio [OR] 2.28; 95% confidence interval [CI] 1.40-3.70; p<0.01), open repair (OR 2.79; 95% CI 1.68-4.63; p<0.01) and general anesthesia (OR 9.16; 95% CI 2.33-36.06; p<0.01). CONCLUSION: Our study revealed that interhospital transfer of patients for urgent repair of AAA was not associated with an increased mortality.

Objetivo: Avaliar a influência da localização geográfica dos doentes com aneurismas da aorta abdominal (AAA) sintomáticos ou rotos (rAAA), na mortalidade. Métodos: Revisão retrospetiva de todos os casos de AAA sintomáticos ou rAAA submetidos a cirurgia numa instituição terciária, entre Janeiro 2011 e Agosto 2017. O outcome primário foi a mortalidade intrahospitalar. Os outcomes secundários foram a admissão em unidade de cuidados intensivos (UCI), duração do internamento na UCI e hospitalar, tipo de cirurgia e anestesia e a apresentação ao fim-de-semana. Os dados foram submetidos a análise univariável e regressão logística. Foi considerado um valor estatisticamente significativo quando o valor de p <0.05. Resultados: 135 doentes foram admitidos com o diagnóstico de AAA sintomático ou rAAA e submetidos a cirurgia, 83 (61.5%) por via endovascular e 52 (38.5%) por via convencional, 30.4% com anestesia local e sedação. 92 doentes (68.1%) foram transferidos de outros hospitais, com uma distância média de 113±88 km. A análise de subgrupos revelou que não existia diferença significativa entre os grupos de doentes transferidos e não transferidos relativamente ao outcome primário (31.5% vs 34.9%, p=0.35), características de base (idade e género), tipo de cirurgia e anestesia, apresentação ao fim-de-semana, admissão na UCI, duração do internamento na UCI e hospitalar. A análise de regressão logística revelou que as variáveis associadas com a mortalidade foram o género feminino (odds ratio [OR] 2.28; 95% intervalo de confiança [IC] 1.40- 3.70; p<0.01), cirurgia convencional (OR 2.79; 95% IC 1.68-4.63; p<0.01) e anestesia geral (OR 9.16; 95% IC 2.33- 36.06; p<0.01). Conclusão: Este estudo revelou que a transferência interhospitalar de doentes para a reparação cirúrgica urgente de AAA não está associada a aumento da mortalidade.

Influence of Interhospital Transfer on Outcomes of Symptomatic and Ruptured Abdominal Aortic Aneurysms.

INTRODUCTION: Symptomatic or ruptured abdominal aortic aneurysms (rAAA) maintains a high mortality index despite technical advances in its treatment. The influence of patients' geographic location on rAAA outcomes, when the rupture occurs or when the AAA becomes symptomatic, has not been a commonly studied issue. Due to the lack of research on this matter, the impact of interhospital transfer on mortality is ambiguous. OBJECTIVE: Evaluate the influence of the geographic location of patients with symptomatic AAA or rAAA on AAA mortality. METHODS: Retrospective review of all cases of symptomatic AAA and rAAA submitted to surgery in a tertiary institution, between January 2011 and August 2017. The main outcome was in-hospital mortality. Secondary outcomes were admission to intensive care unit (ICU), length of ICU and hospital stay, type of repair and anesthesia and weekend presentation. Data was submitted to univariable analysis and logistic regression. Statistical significance was considered if the p value was <0.05. RESULTS: During the defined period of 80 months, a total of 135 patients were admitted with the diagnosis of symptomatic or rAAA and submitted to surgery. Most patients had a ruptured AAA (90.4%, n=122), while symptomatic AAA represented a minority (9.6%, n=13). All patients (91.1% male gender, mean age 74±10 years) were submitted to surgery, 83 (61.5%) by endovascular repair and 52 (38.5%) by open repair, 30.4% with local anesthesia and sedation (n=41), all in the endovascular group. 92 patients (68.1%) were transferred from other hospitals, with a mean distance of 113±88 km. In this cohort, in-hospital mortality was 31.5% in transferred patients and 34.9% in not transferred patients. Subgroup analysis revealed that there were no significant differences between transferred and not transferred patients' groups concerning main outcome (p=0.35), baseline characteristics (age and gender), type of surgery and anesthesia, weekend presentation, ICU admission, length of ICU and hospital stay. Logistic regression analysis revealed that the variables associated with mortality were female gender (odds ratio [OR] 2.28; 95% confidence interval [CI] 1.40-3.70; p<0.01), open repair (OR 2.79; 95% CI 1.68-4.63; p<0.01) and general anesthesia (OR 9.16; 95% CI 2.33-36.06; p<0.01). CONCLUSION: Our study revealed that transfer of patients for urgent repair of AAA was not associated with an increased mortality. The hypothetical increased mortality due to transfer might have been compensated by endovascular treatment and local anesthesia in some cases. Further studies must be carried out, particularly comparing endovascular and open repair in emergency setting.

Infrarenal Abdominal Penetrating Aortic Ulcer, an Atypical Location of a Rare Disease.

INTRODUCTION: Penetrating aortic ulcer (PAU) is classically included in acute aortic syndromes, together with aortic dissection and intramural hematoma. These three disorders are considered different stages of the same disease. PAU is the result of medial degeneration with disruption of the intima, mainly due to atherosclerotic risk factors. Most of them are located on descending thoracic aorta and only a few small series and case reports demonstrate location on infrarenal abdominal aorta. Clinical presentation varies in spectrum, from asymptomatic to fatal aortic rupture. Treatment options include medical therapy, particularly strict blood pressure control, and surgical approach. Nowadays endovascular exclusion is commonly performed, although open surgical reconstruction remains the gold standard. METHODS: Report a case of endovascular repair of an infrarenal abdominal PAU. RESULTS: A 72-year-old man, with hypertension, type 2 diabetes, hypercholesterolemia, lumbar osteoarthrosis, was referred to Vascular Surgery outpatient clinic with the diagnosis of infrarenal abdominal PAU on a Computed Tomography Angiography (CTA). This exam was performed due to chronic lumbar complaints from lumbar osteoarthrosis. The patient denied any other complaint. Physical examination was normal. A thoraco-abdomino-pelvic CTA revealed two sites of PAU in the infrarenal aorta with 10mm and 21mm of depth and associated aortic enlargement of 39mm maximum diameter. This exam revealed an enlargement of the depth of the PAU and the aorta diameter in 2 and 3mm, respectively, in the course of 2 months. An EVAR was performed, in a standard aorto-biiliac fashion. The post-operative period was uneventful and the patient discharged 3 days later. 1 month after the surgery, patient remained asymptomatic and the follow-up CTA demonstrated exclusion of both PAU, no endoleaks and stability of aortic diameter. A long term follow-up should be maintained, as for regular EVAR. CONCLUSION: PAU is a rare clinical entity, with infrarenal abdominal aorta location even scarcer. Asymptomatic patient must be regularly followed and threshold to treatment low, bearing in mind the possible catastrophic evolution of the disease. Endovascular approach should be considered as a first approach, considering the technical feasibility and the comorbidities associated with this elderly population.

Tumour exosome integrins determine organotropic metastasis.

Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6ß4 and α6ß1 were associated with lung metastasis, while exosomal integrin αvß5 was linked to liver metastasis. Targeting the integrins α6ß4 and αvß5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.

Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver.

Pancreatic ductal adenocarcinomas (PDACs) are highly metastatic with poor prognosis, mainly due to delayed detection. We hypothesized that intercellular communication is critical for metastatic progression. Here, we show that PDAC-derived exosomes induce liver pre-metastatic niche formation in naive mice and consequently increase liver metastatic burden. Uptake of PDAC-derived exosomes by Kupffer cells caused transforming growth factor ß secretion and upregulation of fibronectin production by hepatic stellate cells. This fibrotic microenvironment enhanced recruitment of bone marrow-derived macrophages. We found that macrophage migration inhibitory factor (MIF) was highly expressed in PDAC-derived exosomes, and its blockade prevented liver pre-metastatic niche formation and metastasis. Compared with patients whose pancreatic tumours did not progress, MIF was markedly higher in exosomes from stage I PDAC patients who later developed liver metastasis. These findings suggest that exosomal MIF primes the liver for metastasis and may be a prognostic marker for the development of PDAC liver metastasis.

Clinical-scale purification of pluripotent stem cell derivatives for cell-based therapies.

Human pluripotent stem cells (hPSCs) have the potential to revolutionize cell-replacement therapies because of their ability to self renew and differentiate into nearly every cell type in the body. However, safety concerns have delayed the clinical translation of this technology. One cause for this is the capacity that hPSCs have to generate tumors after transplantation. Because of the challenges associated with achieving complete differentiation into clinically relevant cell types, the development of safe and efficient strategies for purifying committed cells is essential for advancing hPSC-based therapies. Several purification strategies have now succeeded in generating non-tumorigenic and homogeneous cell-populations. These techniques typically enrich for cells by either depleting early committed populations from teratoma-initiating hPSCs or by positively selecting cells after differentiation. Here we review the working principles behind separation methods that have facilitated the safe and controlled application of hPSC-derived cells in laboratory settings and pre-clinical research. We underscore the need for improving and integrating purification strategies within differentiation protocols in order to unlock the therapeutic potential of hPSCs.

Integrated platform for production and purification of human pluripotent stem cell-derived neural precursors.

Human pluripotent stem cells (hPSCs) are a promising source of cells for clinical applications, such as transplantation of clinically engineered tissues and organs, and drug discovery programs due to their ability to self-renew and to be differentiated into cells from the three embryonic germ layers. In this study, the differentiation of two hPSC-lines into neural precursors (NPs) was accomplished with more than 80% efficiency, by means of the dual-SMAD inhibition protocol, based on the use of two small molecules (SB431542 and LDN193189) to generate Pax6 and Nestin-positive neural entities. One of the major hurdles related to the in vitro generation of PSC-derived populations is the tumorigenic potential of cells that remain undifferentiated. These remaining hPSCs have the potential to generate teratomas after being transplanted, and may interfere with the outcome of in vitro differentiation protocols. One strategy to tackle this problem is to deplete these "contaminating" cells during the differentiation process. Magnetic activated cell sorting (MACS) was used for the first time for purification of hPSC-derived NPs after the neural commitment stage using anti-Tra-1-60 micro beads for negative selection of the unwanted hPSCs. The depletion had an average efficiency of 80.4 ± 5% and less than 1.5% of Tra-1-60 positive cells were present in the purified populations. After re-plating, the purified neural precursors maintained their phenotype, and the success of the preparative purification with MACS was further confirmed with a decrease of 94.3% in the number of Oct4-positive proliferating hPSC colonies. Thus, the integration of the MACS depletion step with the neural commitment protocol paves the way towards the establishment of a novel bioprocess for production of purified populations of hPSC-derived neural cells for different applications.