Sedimentation field-flow fractionation for rapid phenotypic antimicrobial susceptibility testing: a pilot study.

BACKGROUND: The increase in antibiotic resistance is a major public health issue. The development of rapid antimicrobial susceptibility testing (AST) methods is becoming a priority to ensure early and appropriate antibiotic therapy. OBJECTIVES: To evaluate sedimentation field-flow fractionation (SdFFF) as a method for performing AST in less than 3 h. METHODS: SdFFF is based on the detection of early biophysical changes in bacteria, using a chromatographic-type technology. One hundred clinical Escherichia coli strains were studied. A calibrated bacterial suspension was incubated for 2 h at 37°C in the absence (untreated) or presence (treated) of five antibiotics used at EUCAST breakpoint concentrations. Bacterial suspensions were then injected into the SdFFF machine. For each E. coli isolate, retention times and elution profiles of antibiotic-treated bacteria were compared with retention times and elution profiles of untreated bacteria. Algorithms comparing retention times and elution profiles were used to determine if the strain was susceptible or resistant. Performance evaluation was done according to CLSI and the ISO standard 20776-2:2021 with broth microdilution used as the reference method. RESULTS: AST results from SdFFF were obtained in less than 3 h. SdFFF showed high categorical agreement (99.8%), sensitivity (99.5%) and specificity (100.0%) with broth microdilution. Results for each antimicrobial were also in agreement with the ISO 20776-2 recommendations, with sensitivity and specificity of ≥95.0%. CONCLUSIONS: This study showed that SdFFF can be used as a rapid, accurate and reliable phenotypic AST method with a turnaround time of less than 3 h.

Sedimentation Field-Flow Fractionation: A Diagnostic Tool for Rapid Antimicrobial Susceptibility Testing.

Conventional antimicrobial susceptibility testing (AST) methods require 24-48 h to provide results, creating the need for a probabilistic antibiotic therapy that increases the risk of antibiotic resistance emergence. Consequently, the development of rapid AST methods has become a priority. Over the past decades, sedimentation field-flow fractionation (SdFFF) has demonstrated high sensitivity in early monitoring of induced biological events in eukaryotic cell populations. This proof-of-concept study aimed at investigating SdFFF for the rapid assessment of bacterial susceptibility to antibiotics. Three bacterial species were included (Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa) with two panels of antibiotics tailored to each bacterial species. The results demonstrate that SdFFF, when used in "Hyperlayer" elution mode, enables monitoring of antibiotic-induced morphological changes. The percentage variation of the retention factor (PΔR) was used to quantify the biological effect of antibiotics on bacteria with the establishment of a threshold value of 16.8% to differentiate susceptible and resistant strains. The results obtained with SdFFF were compared to that of the AST reference method, and a categorical agreement of 100% was observed. Overall, this study demonstrates the potential of SdFFF as a rapid method for the determination of antibiotic susceptibility or resistance since it is able to provide results within a shorter time frame than that needed for conventional methods (3-4 h vs 16-24 h, respectively), enabling earlier targeted antibiotic therapy. Further research and validation are necessary to establish the effectiveness and reliability of SdFFF in clinical settings.

Detection of Glycosylated Markers From Cancer Stem Cells With ColoSTEM Dx Kit for Earlier Prediction of Colon Cancer Aggressiveness.

Nowadays, colon cancer prognosis still difficult to predict, especially in the early stages. Recurrences remain elevated, even in the early stages after curative surgery. Carcidiag Biotechnologies has developed an immunohistochemistry (IHC) kit called ColoSTEM Dx, based on a MIX of biotinylated plant lectins that specifically detects colon cancer stem cells (CSCs) through glycan patterns that they specifically (over)express. A retrospective clinical study was carried out on tumor tissues from 208 non-chemotherapeutic-treated and 21 chemotherapeutic-treated patients with colon cancer, which were stained by IHC with the MIX. Clinical performances of the kit were determined, and prognostic and predictive values were evaluated. With 78.3% and 70.6% of diagnostic sensitivity and specificity respectively, our kit shows great clinical performances. Moreover, patient prognosis is significantly poorer when the MIX staining is "High" compared to "Low", especially at 5-years of overall survival and for early stages. The ColoSTEM Dx kit allows an earlier and a more precise determination of patients' outcome. Thus, it affords an innovating clinical tool for predicting tumor aggressiveness earlier and determining prognosis value regarding therapeutic response in colon cancer patients.

A Label-Free Cell Sorting Approach to Highlight the Impact of Intratumoral Cellular Heterogeneity and Cancer Stem Cells on Response to Therapies.

Cancer stem cells play a crucial role in tumor initiation, metastasis, and resistance to treatment. Cellular heterogeneity and plasticity complicate the isolation of cancer stem cells. The impact of intra-tumor cellular heterogeneity using a label-free approach remains understudied in the context of treatment resistance. Here, we use the sedimentation field-flow fractionation technique to separate, without labeling, cell subpopulations of colorectal cancer cell lines and primary cultures according to their biophysical properties. One of the three sorted cell subpopulations exhibits characteristics of cancer stem cells, including high tumorigenicity in vivo and a higher frequency of tumor-initiating cells compared to the other subpopulations. Due to its chemoresistance, two- and three-dimensional in vitro chemosensitivity assays highlight the therapeutic relevance of this cancer stem cell subpopulation. Thus, our results reveal the major implication of intra-tumor cellular heterogeneity, including cancer stem cells in treatment resistance, thanks to our label-free cell sorting approach. This approach enables-by breaking down the tumor-the study the individualized response of each sorted tumor cell subpopulation and to identify chemoresistance, thus offering new perspectives for personalized therapy.

Nucleoside-Derived Low-Molecular-Weight Gelators as a Synthetic Microenvironment for 3D Cell Culture.

For the last few decades, many efforts have been made in developing cell culture methods in order to overcome the biological limitations of the conventional two-dimensional culture. This paradigm shift is driven by a large amount of new hydrogel-based systems for three-dimensional culture, among other systems, since they are known to mimic some living tissue properties. One class of hydrogel precursors has received interest in the field of biomaterials, low-molecular-weight gelators (LMWGs). In comparison to polymer gels, LMWG gels are formed by weak interactions upon an external trigger between the molecular subunits, giving them the ability to reverse the gelation, thus showing potential for many applications of practical interest. This study presents the use of the nucleoside derivative subclass of LMWGs, which are glyco-nucleo-bola-amphiphiles, as a proof of concept of a 3D cell culture scaffold. Physicochemical characterization was performed in order to reach the optimal features to fulfill the requirements of the cell culture microenvironment, in terms of the mechanical properties, architecture, molecular diffusion, porosity, and experimental practicality. The retained conditions were tested by culturing glioblastoma cells for over a month. The cell viability, proliferation, and spatial organization showed during the experiments demonstrate the proof of concept of nucleoside-derived LMWGs as a soft 3D cell culture scaffold. One of the hydrogels tested permits cell proliferation and spheroidal organization over the entire culture time. These systems offer many advantages as they consume very few matters within the optimal range of viscoelasticity for cell culture, and the thermoreversibility of these hydrogels permits their use with few instruments. The LMWG-based scaffold for the 3D cell culture presented in this study unlocked the ability to grow spheroids from patient cells to reach personalized therapies by dramatically reducing the variability of the lattice used.

Characterization of Glioblastoma Cancer Stem Cells Sorted by Sedimentation Field-Flow Fractionation Using an Ultrahigh-Frequency Range Dielectrophoresis Biosensor.

Cancer stem cells (CSCs) appear to be an essential target for cancer therapies, in particular, in brain tumors such as Glioblastoma. Nevertheless, their isolation is made difficult by their low content in culture or tumors (<5% of the tumor mass) and is essentially based on the use of fluorescent or magnetic labeling techniques, increasing the risk of differentiation induction. The use of label-free separation methods such as sedimentation field-flow fractionation (SdFFF) is promising, but it becomes necessary to consider a coupling with a detection and characterization method for future identification and purification of CSCs from patient-derived tumors. In this study, we demonstrate for the first time the capability of using an ultrahigh-frequency range dielectrophoresis fluidic biosensor as a detector. This implies an important methodological adaptation of SdFFF cell sorting by the use of a new compatible carrier liquid DEP buffer (DEP-B). After SdFFF sorting, subpopulations derived from U87-MG and LN18 cell lines undergo biological characterization, demonstrating that using DEP-B as a carrier liquid, we sorted by SdFFF subpopulations with specific differentiation characteristics: F1 = differentiated cells/F2 = CSCs. These subpopulations presented high-frequency crossover (HFC) values similar to those measured for standard differentiated (around 110 MHz) and CSC (around 80 MHz) populations. This coupling appeared as a promising solution for the development of an online integration of these two complementary label-free separation/detection technologies.

Autophagy inhibition reinforces stemness together with exit from dormancy of polydisperse glioblastoma stem cells.

Therapeutic resistance and infiltrative capacities justify the aggressiveness of glioblastoma. This is due to cellular heterogeneity, especially the presence of stemness-related cells, i.e. Cancer Stem Cells (CSC). Previous studies focused on autophagy and its role in CSCs maintenance; these studies gave conflicting results as they reported either sustaining or disruptive effects. In the present work, we silenced two autophagy related genes -either Beclin1 or ATG5- by shRNA and we explored the ensuing consequences on CSCs markers' expression and functionalities. Our results showed that the down regulation of autophagy led to enhancement in expression of CSCs markers, while proliferation and clonogenicity were boosted. Temozolomide (TMZ) treatment failed to induce apoptotic death in shBeclin1-transfected cells, contrary to control. We optimized the cellular subset analysis with the use of Sedimentation Field Flow Fractionation, a biological event monitoring- and cell sorting-dedicated technique. Fractograms of both shBeclin1 and shATG5 cells exhibited a shift of elution peak as compared with control cells, showing cellular dispersion and intrinsic sub-fraction modifications. The classical stemness fraction (i.e. F3) highlighted data obtained with the overall cellular population, exhibiting enhancement of stemness markers and escape from dormancy. Our results contributed to illustrate CSCs polydispersity and to show how these cells develop capacity to bypass autophagy inhibition, thanks to their acute adaptability and plasticity.

Autophagy and Extracellular Vesicles, Connected to rabGTPase Family, Support Aggressiveness in Cancer Stem Cells.

Even though cancers have been widely studied and real advances in therapeutic care have been made in the last few decades, relapses are still frequently observed, often due to therapeutic resistance. Cancer Stem Cells (CSCs) are, in part, responsible for this resistance. They are able to survive harsh conditions such as hypoxia or nutrient deprivation. Autophagy and Extracellular Vesicles (EVs) secretion are cellular processes that help CSC survival. Autophagy is a recycling process and EVs secretion is essential for cell-to-cell communication. Their roles in stemness maintenance have been well described. A common pathway involved in these processes is vesicular trafficking, and subsequently, regulation by Rab GTPases. In this review, we analyze the role played by Rab GTPases in stemness status, either directly or through their regulation of autophagy and EVs secretion.

The Role of Cancer Stem Cells in Colorectal Cancer: From the Basics to Novel Clinical Trials.

The treatment options available for colorectal cancer (CRC) have increased over the years and have significantly improved the overall survival of CRC patients. However, the response rate for CRC patients with metastatic disease remains low and decreases with subsequent lines of therapy. The clinical management of patients with metastatic CRC (mCRC) presents a unique challenge in balancing the benefits and harms while considering disease progression, treatment-related toxicities, drug resistance and the patient's overall quality of life. Despite the initial success of therapy, the development of drug resistance can lead to therapy failure and relapse in cancer patients, which can be attributed to the cancer stem cells (CSCs). Thus, colorectal CSCs (CCSCs) contribute to therapy resistance but also to tumor initiation and metastasis development, making them attractive potential targets for the treatment of CRC. This review presents the available CCSC isolation methods, the clinical relevance of these CCSCs, the mechanisms of drug resistance associated with CCSCs and the ongoing clinical trials targeting these CCSCs. Novel therapeutic strategies are needed to effectively eradicate both tumor growth and metastasis, while taking into account the tumor microenvironment (TME) which plays a key role in tumor cell plasticity.

Intraperitoneal Chemotherapy for Peritoneal Metastases: Technical Innovations, Preclinical and Clinical Advances and Future Perspectives.

(1) Background: Tumors of the peritoneal serosa are called peritoneal carcinosis. Their origin may be primary by primitive involvement of the peritoneum (peritoneal pseudomyxoma, peritoneal mesothelioma, etc.). This damage to the peritoneum can also be a consequence of the dissipation of cancers-in particular, digestive (stomach, pancreas, colorectal, appendix) and gynecological (ovaries) ones in the form of metastases. The aim of the treatment is a maximal reduction of the macroscopic disease called "cytoreduction" in combination with hyperthermic intra-abdominal chemotherapy to treat residual microscopic lesions. (2) Methods: In this narrative review, we fundamentally synthetize the evolution of this process over time and its impact on clinical applications. (3) Results: Over the last past decade, different evolutions concerning both delivery modes and conditions concerning hyperthermic intra-abdominal chemotherapy have been realized. (4) Conclusion: The final objective of these evolutions is the improvement of the global and recurrence-free survival of primary and secondary malignant peritoneal pathologies. However, more large randomized controlled trials are needed to demonstrate the efficacy of such treatments with the help of molecular biology and genetics.

Biomaterials for Three-Dimensional Cell Culture: From Applications in Oncology to Nanotechnology.

Three-dimensional cell culture has revolutionized cellular biology research and opened the door to novel discoveries in terms of cellular behavior and response to microenvironment stimuli. Different types of 3D culture exist today, including hydrogel scaffold-based models, which possess a complex structure mimicking the extracellular matrix. These hydrogels can be made of polymers (natural or synthetic) or low-molecular weight gelators that, via the supramolecular assembly of molecules, allow the production of a reproducible hydrogel with tunable mechanical properties. When cancer cells are grown in this type of hydrogel, they develop into multicellular tumor spheroids (MCTS). Three-dimensional (3D) cancer culture combined with a complex microenvironment that consists of a platform to study tumor development and also to assess the toxicity of physico-chemical entities such as ions, molecules or particles. With the emergence of nanoparticles of different origins and natures, implementing a reproducible in vitro model that consists of a bio-indicator for nano-toxicity assays is inevitable. However, the maneuver process of such a bio-indicator requires the implementation of a repeatable system that undergoes an exhaustive follow-up. Hence, the biggest challenge in this matter is the reproducibility of the MCTS and the associated full-scale characterization of this system's components.

Overexpression of sortilin is associated with 5-FU resistance and poor prognosis in colorectal cancer.

Colorectal cancer (CRC) is the third most common cancer worldwide. Even if 5-fluorouracil (5-FU) is used as the first-line chemotherapeutic drug, responsiveness is only 20-30%. Acquired resistance to 5-FU contributes to both poor patient prognosis and relapse, emphasizing the need to identify biomarkers. Sortilin, a vacuolar protein sorting 10 protein (Vps10p), implicated in protein trafficking, is over expressed in CRC cell lines cultured 72 hours in presence of 5-FU. This overexpression was also observed in 5-FU-resistant cells derived from these cell lines as well as in CRC primary cultures (or patients derived cell lines). A significantly higher expression of sortilin was observed in vivo, in 5-FU-treated tumours engrafted in Nude mice, as compared with non-treated tumour. A study of transcriptional regulation allowed identifying a decrease in ATF3 expression, as an explanation of sortilin overexpression following 5-FU treatment. In silico analysis revealed SORT1 expression correlation with poor prognosis. Moreover, sortilin expression was found to be positively correlated with CRC tumour grades. Collectively, our findings identify sortilin as a potential biomarker of 5-FU resistance associated with poor clinical outcomes and aggressiveness in CRC. As a new prognostic factor, sortilin expression could be used to fight against CRC.

Self-Assembly of Nucleoside-Derived Low-Molecular-Weight Gelators: A Thermodynamics and Kinetics Study on Different Length Scales.

Biocompatible materials are of paramount importance in numerous fields. Unlike chemically bridge polymer-based hydrogels, low-molecular-weight gelators can form a reversible hydrogel as their structures rely on noncovalent interaction. Although many applications with this type of hydrogel can be envisioned, we still lack their understanding due to the complexity of their self-assembly process and the difficulty in predicting their behaviors (transition temperature, gelation kinetics, the impact of solvent, etc.). In this study, we extend the investigations of a series of nucleoside-derived gelators, which only differ by subtle chemical modifications. Using a multitechnique approach, we determined their thermodynamic and kinetic features on various scale (molecular to macro) in different conditions. Monitored at the supramolecular level by circular dichroism as well as macroscopic scales by rheology and turbidimetry, we found out that the sol-gel and gel-sol transitions are greatly dependent on the concentration and on the mechanisms that are probed. Self-assembly kinetics depends on hydrogel molecules and is modulated by temperature and solvent. This fundamental study provides insight on the impact of some parameters on the gelation process, such as concentration, cooling rate, and the nature of the solvent.

A New Label-Free Approach to Glioblastoma Cancer Stem Cell Sorting and Detection.

Cancer stem cells (CSCs) play critical roles in cancer, making them important targets for new diagnostic and therapeutic approaches. Since CSCs are heterogeneous and not abundant in tumors, and few specific markers for these cells currently exist, new methods to isolate and characterize them are required. To address this issue, we developed a new label-free methodology to isolate, enrich, and identify CSCs from an heterogeneous tumor cell subpopulation using a cell sorting method (sedimentation field flow fractionation, SdFFF) and a biosensor as a detector. Enrichment was optimized using an original protocol and U87-MG glioblastoma cells cultured in a normal (N) or defined (D) medium (± fetal bovine serum, FBS) under normoxic (N, pO2 = 20%) or hypoxic (H, pO2 < 2%) conditions to obtain four cell populations: NN, NH, DN, and DH. After elution of CSCs via SdFFF using the hyperlayer mode (inertial elution mode for micrometer-sized species), we isolated eight subpopulations with distinct CSC contents based on phenotypical and functional properties, ranging from NN F1 with a lower CSC content to DH F3 with a higher CSC content. Reflecting biological differences, the intrinsic intracellular dielectric permittivity increased from NN to DH conditions. The largest difference in electromagnetic signature was observed between NN F1 and DH F3, in which the CSC content was lowest and highest, respectively. The results demonstrate that microwave dielectric spectroscopy can be used to reliably and efficiently distinguish stem cell characteristics. This new instrumental and methodological approach is an important innovation that allows both enrichment and detection of CSCs, opening the door to novel diagnostic and therapeutic approaches.

E-cadherin: A potential biomarker of colorectal cancer prognosis.

Colorectal cancer (CRC) is a common and lethal disease. It is the third most common type of cancer in the world, behind lung and breast cancer, with almost 1.4 million new cases diagnosed in 2012. The risk of developing CRC is influenced by environmental and genetic factors. Adenocarcinomas comprise the vast majority (98%) of CRCs. A patient's likelihood of survival is associated with the tumor stage at the time of diagnosis. With regular screening, CRC can be identified early, when treatment is the most effective. However, CRC is typically asymptomatic until the advanced stages. The combination of the absence of symptoms and current screening methodology results in a significant number of patients being diagnosed in advanced stages. The purpose of the present review is to discuss and summarize the biomarkers linked to CRC progression, particularly the controversial E-cadherin, which is a calcium-dependent cell-cell adhesion molecule involved in the mesenchymal-epithelial transition.

Isolation of Astrocytes Displaying Myofibroblast Properties and Present in Multiple Sclerosis Lesions.

A wide heterogeneity of lesions can affect the central nervous system (CNS). In all situations where neurons are damaged, including multiple sclerosis (MS), a common reactive astrocytosis is present. Sedimentation field-flow fractionation (SdFFF) was used to sort astrocyte subpopulations. After SdFFF elution, cells, prepared from rat newborn cortex, were cultured and analyzed by immunocytofluorescence for glial fibrillary acidic protein (GFAP) and α-smooth muscle (SM) actin (a specific marker for myofibroblasts) expression. Cell contractile capacity was studied. Samples from patients with MS were also analyzed. Three main fractions (F1, F2, and F3) were isolated and compared with the total eluted population (TP). TP, F1, F2, and F3, contained respectively 74, 96, 12, and 98% of GFAP expressing astrocytes. In F3, astrocytes only expressed GFAP while in F1, astrocytes expressed both GFAP and α-SM actin. In F2 and TP, α-SM actin expression was barely detected. F3-derived cells showed higher contractile capacities compared with F1-derived cells. In one specific case of MS known as Baló's concentric MS, astrocytes expressing both GFAP and α-SM actin were detected. Using SdFFF, a population of astrocytes presenting myofibroblast properties was isolated. This subpopulation of astrocytes was also observed in a MS sample suggesting that it could be involved in lesion formation and remodeling during CNS pathologies.

The extracellular domain of E cadherin linked to invasiveness in colorectal cancer: a new resistance and relapses monitoring serum-bio marker?

PURPOSE: Multiple studies have attempted to demonstrate the interest of the cell adhesion marker, E cadherin, as a diagnostic and prognosis marker in colorectal cancer (CRC). However, it was considered non specific. MATERIALS AND METHODS: Studies were carried out with CRC cell lines and patients' cohort operated for CRC. The expression of E cadherin was studied after 5 fluorouracil (5FU) treatment and correlated to CRC relapse, chemo-resistance and survival. RESULTS: In CRC cell lines derived from high tumor stages, extracellular domain of E cadherin expression decreased after 5FU treatment whereas it increased in supernatants. Interestingly, only specific cleaved forms at 55 kDa of E cadherin were detected in supernatants. In CRC surgical patients, more importantly concerning extracellular E cadherin domain, a decreased expression was observed in tissues in function of CRC stages whereas an increased expression was found in sera. Moreover, there is an increasing trend of survival with weak serum E cadherin secretion, reinforcing the implication of this protein in CRC evolution. DISCUSSION: The extracellular domain can be defined as a 5FU resistance marker and allow CRC monitoring.

KLRC3, a Natural Killer receptor gene, is a key factor involved in glioblastoma tumourigenesis and aggressiveness.

Glioblastoma is the most lethal brain tumour with a poor prognosis. Cancer stem cells (CSC) were proposed to be the most aggressive cells allowing brain tumour recurrence and aggressiveness. Current challenge is to determine CSC signature to characterize these cells and to develop new therapeutics. In a previous work, we achieved a screening of glycosylation-related genes to characterize specific genes involved in CSC maintenance. Three genes named CHI3L1, KLRC3 and PRUNE2 were found overexpressed in glioblastoma undifferentiated cells (related to CSC) compared to the differentiated ones. The comparison of their roles suggest that KLRC3 gene coding for NKG2E, a protein initially identified in NK cells, is more important than both two other genes in glioblastomas aggressiveness. Indeed, KLRC3 silencing decreased self-renewal capacity, invasion, proliferation, radioresistance and tumourigenicity of U87-MG glioblastoma cell line. For the first time we report that KLRC3 gene expression is linked to glioblastoma aggressiveness and could be a new potential therapeutic target to attenuate glioblastoma.

TrkB-containing exosomes promote the transfer of glioblastoma aggressiveness to YKL-40-inactivated glioblastoma cells.

The neurotrophin receptors are known to promote growth and proliferation of glioblastoma cells. Their functions in spreading glioblastoma cell aggressiveness to the microenvironment through exosome release from glioblastoma cells are unknown. Considering previous reports demonstrating that YKL-40 expression is associated with undifferentiated glioblastoma cancer stem cells, we used YKL-40-silenced cells to modulate the U87-MG differentiated state and their biological aggressiveness. Herein, we demonstrated a relationship between neurotrophin-receptors and YKL-40 expression in undifferentiated cells. Differential functions of cells and derived-exosomes were evidenced according to neurotrophin receptor content and differentiated cell state by comparison with control pLKO cells. YKL-40 silencing of glioblastoma cells impairs proliferation, neurosphere formation, and their ability to induce endothelial cell (HBMEC) migration. The modulation of differentiated cell state in YKL-40-silenced cells induces a decrease of TrkB, sortilin and p75NTR cellular expressions, associated with a low-aggressiveness phenotype. Interestingly, TrkB expressed in exosomes derived from control cells was undetectable in exosomes from YKL-40 -silenced cells. The transfer of TrkB-containing exosomes in YKL-40-silenced cells contributed to restore cell proliferation and promote endothelial cell activation. Interestingly, in U87 MG xenografted mice, TrkB-depleted exosomes from YKL-40-silenced cells inhibited tumor growth in vivo. These data highlight that TrkB-containing exosomes play a key role in the control of glioblastoma progression and aggressiveness. Furthermore, TrkB expression was detected in exosomes isolated from plasma of glioblastoma patients, suggesting that this receptor may be considered as a new biomarker for glioblastoma diagnosis.

New Method for Sorting Endothelial and Neural Progenitors from Human Induced Pluripotent Stem Cells by Sedimentation Field Flow Fractionation.

Human induced pluripotent stem cells (hiPSc) are a very useful solution to create and observe the behavior of specific and usually inaccessible cells, such as human motor neurons. Obtained from a patient biopsy by reprograming dermal fibroblasts (DF), hiPSc present the same properties as embryonic stem cells and can generate any cell type after several weeks of differentiation. Today, there are numerus protocols which aim to control hiPSC differentiation. The principal challenge is to obtain a sufficiently enriched specific cell population to study disease pathophysiology and to provide a good model for further investigation and drug screening. The differentiation process is very costly and time-consuming, because many specific factors and different culture media must be used. In this study, we used Sedimentation Field Flow Fractionation (SdFFF) to prepare enriched populations derived from hiPSc after only 10 days of culture in a classical medium. Based on phenotypic and proteomic characterization, "hyperlayer" elution resulted in a fraction expressing markers of endothelial progenitors while another fraction expressed markers of neural progenitors. The isolation of subpopulations representing various differentiation lineages is of major interest for the production of specialized, cell-enriched fractions and in the preparation of increasingly complex models for the development of new therapeutic tools.