New Method for Sorting Endothelial and Neural Progenitors from

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A New Rapid Method for Sorting Endothelial and Neural Progenitors from Human Induced Pluripotent Stem Cells by Sedimentation Field Flow Fractionation. Pierre Antoine Faye, Nicolas Vedrenne, Miguel Angel de la Cruz Morcillo, Claire Cécile Barrot, Laurence Richard, Sylvie Bourthoumieu, Franck Sturtz, Benoit Funalot, Anne-Sophie Lia, and Serge Battu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00704 • Publication Date (Web): 06 Jun 2016 Downloaded from http://pubs.acs.org on June 8, 2016

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Analytical Chemistry

A New Rapid Method for Sorting Endothelial and Neural Progenitors from Human Induced Pluripotent Stem Cells by Sedimentation Field Flow Fractionation. Pierre-Antoine Faye(1), Nicolas Vedrenne(2), Miguel A. De la Cruz-Morcillo(3), Claire-Cécile Barrot(1,4), Laurence Richard(1,5), Sylvie Bourthoumieu(1,6), Franck Sturtz(1,4), Benoît Funalot(7,8), Anne-Sophie Lia(1,4) and Serge Battu(2*).

1- Univ. Limoges. Maintenance Myélinique et Neuropathies Périphériques, EA6309, F-87000 Limoges, France. 2- Univ. Limoges. Homéostasie Cellulaire et Pathologies, Laboratoire de Bromatologie EA 3842, F87000 Limoges, France. 3- Univ. Limoges. Homéostasie Cellulaire et Pathologies, EA 3842, F-87000 Limoges, France. 4- CHU Limoges. Service de Biochimie et Génétique Moléculaire, F-87000 Limoges, France. 5- CHU Limoges. Service de Neurologie, F-87000 Limoges, France. 6- CHU Limoges. Service de Cytogénétique, F-87000 Limoges, France. 7- CHU Henri-Mondor. Département de Génétique, F-94000 Créteil, France. 8- Inserm U955-E10. Université Paris-Est-Créteil, F-94000 Créteil, France. * Corresponding author: Pr Serge Battu, Tel: +33 5 55 43 59 79, Email: [email protected]

Key words: Human induced pluripotent stem cells, cell sorting, sedimentation field flow fractionation, neural progenitors, endothelial progenitors.

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Abstract 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 provide a good model for further investigation and drug screening. The differentiation process is very costly and timeconsuming 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.


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Introduction Human induced pluripotent stem cells (hiPSc) have the same properties as human embryonic stem cells (hESc), which are self-renewable and have the ability to differentiate into all cell types that make up an adult organism1. In the absence of growth factors that maintain their pluripotent properties, hiPSc differentiate into the three embryonic germ layers: ectoderm, endoderm, and mesoderm, similarly to hESc. HiPSc are obtained by genetic reprograming of differentiated adult cells (skin dermal fibroblasts) using non-integrative plasmids (Oct4, Sox2, Klf4, l-Myc)2, 3. HiPSc have been used to model various neurological disorders such as Parkinson’s disease4, Alzheimer’s disease5 or Charcot Marie Tooth disease (CMT)6, and to test the efficiency of candidate drugs at various scales, such as by High-Throughput Screening7, 8. HiPSc could also become an excellent tool for cell therapy and the regeneration and grafting of whole organs9. There are many different protocols to derive most specialized cells, such as neural cells10, cardiomyocytes11, retinal pigmentary epithelium12, etc. Nevertheless, it is still difficult to control the differentiation process, especially for the preparation of complex cell models because of the heterogeneity of the final culture, and obtain reliable and reproducible results in pharmaceutical screening13. It is crucial to develop new methods to rapidly obtain pure and intact populations of hiPSc-derived progenitors to improve progress in human health. Various techniques are available for cell separation and characterization, such as fluorescent or magnetic-activated cell sorting (FACS or MACS), which take advantage of specific antigen expression. However, phenotypic characterization is controversial and the specific antibody labeling required for these techniques can by itself induce cell modifications (including cell differentiation)14. It is essential to develop alternative methods to enrich hiPSc-derived progenitors without labeling to avoid interference with further cell use (culture, graft, differentiation, etc.). 3 ACS Paragon Plus Environment


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Methods based on intrinsic biophysical properties (size, density, etc.) such as field flow fractionation (FFF), are of great interest. Among FFF techniques, Sedimentation FFF (SdFFF) is a gentle, non-invasive, tagless cell sorting method15,

16

. These advantages are based on

substantially limiting cell-solid phase interactions by the use of 1) an empty ribbon-like channel without a stationary phase; and 2) the "hyperlayer" elution mode, a size/density driven separation mechanism17, 18, 19, 20, 21, 22, 23, 24. The principle of cell separation is based on physical criteria such as size, density, shape, and rigidity15,

16, 17, 19

, and depends on the

differential elution of species submitted to the combined action of 1) a parabolic profile generated by flowing a mobile phase through the channel; and 2) an external field applied perpendicularly to the flow direction15, 16, 17, 19. In SdFFF, a multigravitational external field is generated by rotation of the separation channel in a rotor basket, constituting one of the most complex devices used in FFF separation25. Since our pioneering report concerning mouse embryonic stem cells26, we have demonstrated the utility of SdFFF in preparing enriched and sterile populations of normal and cancer stem cells27, 28, 29, 30. The aim of this study was to evaluate the capacity of SdFFF to isolate specific early enriched populations of hiPSc-derived progenitors. Fractions were characterized by proteomic studies, showing that SdFFF can sort enriched hiPSc-derived progenitors from the neural and endothelial lineages. This could be achieved only 10 days after spontaneous Embryoid Bodies (EB) differentiation, without specific factors, in a basic medium, in contrast to the canonical way, which requires more time, selective culture medium, and differentiation factors.


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Experimental section The overall protocol for hiPSc preparation, characterization, differentiation, and preparation of neural progenitors is presented in figure 1. Human iPSc generation and culture conditions Dermal fibroblasts (DF). After obtaining informed consent from patients (“Centre de Référence National des Neuropathies Périphériques Rares, CHU Service de Neurologie,” Limoges, France), skin biopsies were performed and used to obtain a primary culture of fibroblasts by the explant technique. Explants of 1 mm3 were cultured in CHANG Medium D (Irvine Scientific, Santa Ana, USA) for three days. Then the proportion of CHANG medium D (Irvine Scientific) was reduced by mixing with RPMI-1640 Medium (Life Technologies, Carlsbad, CA, USA) at a 1:1 ratio and the cells were cultured for two days. The cells were cultured thereafter in a 1:4 mix of CHANG to RPMI medium. All media were supplemented with 10% fetal calf serum (FCS) (Life Technologies), 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies). Tissue culture flasks were maintained at 37 °C in a watersaturated atmosphere and 5% CO2. After two weeks, the explants were removed from the culture flasks and the DF were ready for hiPSc reprograming. Prior to hiPSc generation, human fibroblasts were tested for HVB, HVC, and HIV (CHU Virology department, Limoges, France) and verified to be mycoplasma-free (MycoAlert™, Lonza, Walkersville, MD, USA).



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Figure 1: Protocol for hiPSc preparation, characterization, and differentiation to obtain neural progenitors by the canonical way vs SdFFF elution.


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HiPSc generation. Three non-integrative plasmids (Plasmid #6 pCXLE-hOCT3/4 shp53-F Addgene, Plasmid #7 pCXLE-hSK Addgene, Plasmid #8 pCXLE-hUL Addgene) at 1 µg/mL were used to reprogram fibroblasts into hiPSc by using Nucleofector II (Amaxa, Lonza) according to the iStem procedure (INSERM/UEVE UMR 861, AFM, Genopole, Evry, France). Immediately after nucleofection, 100,000 cells were seeded in each well of a six-well tissue culture plate, which had previously been seeded with mitomycin C-treated BJ-1 cells (iStem construct). The cells were cultured in DMEM GlutaMAX (Life Technologies), supplemented with 10% fetal bovine serum (Life Technologies) and 1X MEM non-essential amino acids (Life Technologies). Tissue culture plates were maintained at 37 °C in a watersaturated atmosphere and 5% CO2. At day 1, the medium was changed and supplemented with 10 UI/mL gentamycin (Life Technologies). At day 4, hiPSc medium was added: KODMEM (Life Technologies), supplemented with 20% KnockOut Serum Replacement (KSR) (Life Technologies), 1X MEM non-essential amino acids, 2 mM Glutamax (Life Technologies), 50 µM β-mercaptoethanol (Life Technologies), and 10 UI/mL gentamycin (Life Technologies). The medium was supplemented with 10 ng/mL FGF2 (Peprotech, Rocky Hill, NJ, USA), 2 µM SB431542 (Tocris Bioscience, Minneapolis, MN, USA), 0.5µM PD0325901 (Miltenyi Biotech, Bergisch Gladbach, Germany), and 500 µM valproate (SigmaAldrich, Saint-Quentin Fallavier, France) for 10 days and changed every 2 days. The hiPSc colonies were repicked approximately 2-5 weeks post nucleofection and were maintained in culture for six months with care. The same supplemented hiPSc medium was used to feed the cells every two days. The cells were used at passage 20 to perform controls and characterize the differentiation of the hiPSc.



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hiPSc culture, EB formation, and spontaneous differentiation. Colonies were cultivated on 0.1% gelatin (Sigma-Aldrich) in dishes previously seeded with mitomycin C-treated Mouse Embryonic Fibroblasts (MEF) (feeder layer; TebuBio, Le Perray-en-Yvelines, France). HiPSc were cleaned every day by removing differentiated cells with a needle. Medium was replaced daily by fresh hiPSc medium supplemented with 10 ng/mL FGF2. HiPSc colonies were passaged weekly by cutting colonies into large squares using a needle. EB were plated on dishes coated with 0.1% gelatin (Sigma-Aldrich) and cultured in basic medium, which is the hiPSc medium as previously described without FGF2. After 10 days, the cells were removed and isolated using Accutase (Sigma-Aldrich). Accutase was removed by centrifugation, and DPBS/basic medium (40/60 v/v) was added to the pellet to obtain a suspension with no aggregates and ready for SdFFF.

SdFFF device and cell elution conditions. The SdFFF separation device used in this study was based on those previously described31. The apparatus was composed of two 880 × 55 × 2 mm polystyrene plates, separated by a mylar® spacer in which the channel was carved. Channel dimensions were 781 mm × 10 mm × 0.125 mm with two 70 mm V-shaped ends with a measured total void volume of 1133 ± 0.70 µL (n > 6). The channel rotor axis distance was measured to be r = 13.80 cm. Sedimentation fields are expressed in units of gravity, 1g = 980 cm/s2. Cleaning and decontamination procedures and the control of rotation speed, as well as chromatographic and acquisition devices have been previously described15, 31. The optimal elution conditions were experimentally determined and were: flow injection of 100 µL hiPSc derived progenitor suspension (2 x 106 cells/mL); flow rate: 0.60 mL/min; mobile phase: sterile PBS, pH 7.4; external multi-gravitational field strength: 40.00 ± 0.02 g; spectrophotometric detection at 254 nm. The inlet tubing connecting the injection device to the separation channel (via the rotating seals) is directly screwed into the accumulation wall. Fractions were collected as follows: total population (TP): 3 min 00 s to 11 min 30 s; fraction 8 ACS Paragon Plus Environment

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1 (F1): 3 min 00 s to 5 min 30 s; fraction 2 (F2): 5 min 30 s to 7 min 45 s; fraction 3 (F3): 7 min 45 s to 11 min 30 s. Successive SdFFF fraction collections were performed (6 to 9) to obtain a sufficient quantity of cells for further biological and biophysical characterization. Size measurement using a Coulter Counter31.

Coulter Counter. A 256 channel Multisizer II Coulter Counter (Beckman Coulter, Fullerton, CA) was used to determine the mean diameter and size distribution of cells from the hiPSc subpopulations. The cells were diluted in Isoton® to a final volume of 15 mL. The counting conditions were: 500 µL sample volume, cumulative results of three successive assays. Results are given as the mean ± SD for three different experiments.

Protein analysis, mass spectrometry (MS), and peptide identification. Chemical products were purchased from GE Healthcare for sodium dodecyl sulfate (SDS)-PAGE analysis and from Sigma-Aldrich for mass spectrometry analysis. After SdFFF cell sorting, the cells were cultured during one week in 6 well plates (1×105 cells per well). The cells from each fraction were lysed (5 min on ice) in the culture plate by RIPA lysis buffer (1% NP-40, 0.1%

SDS,

0.5%

desoxy-cholate

sodium

in

PBS)

supplemented

with

1

mM

phenylmethylsulfonyl fluoride. The suspension was sonicated for 1 min (a pulse of 40 Hz every 20 sec) to degrade chromosomal DNA and centrifuged at 14,000 x g for 10 min. Supernatants were collected and the protein concentration determined using the Bradford protein concentration assay. Proteins were resolved on a 10% SDS-polyacrylamide gel and visualized by Colloïdal CBB G250 staining. The gel profiles of the different fractions were compared, the protein zones of interest excised, and protein digestion performed using sequencing grade modified trypsin (Promega, Charbonnières, France) as previously described31. After gel digestion, the peptides were analyzed by nano LC-MS/MS using an LC 9 ACS Paragon Plus Environment


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Packings system (Dionex, Amsterdam, The Nederland) coupled with a Triple TOF 5600+ (Sciex, Montréal, Canada). The Triple TOF was operated in the information-dependent acquisition mode using Analyst 1.7 TF software (Sciex). For database searching, the MS/MS data were used to query the Swissprot database using Mascot software (version 2.2, Matrix Science, England) with the following criteria: human as taxonomy category, 0.5 Da for peptide and fragment mass tolerances, one missed trypsin cleavage site allowed, carbamidomethylation of cysteine residues (from iodoacetamide exposure) and methionine oxidation as variable modifications. Protein identification was established with one peptide match with an ion score above 50 or at least two peptide matches with an ion score above 25 at a 95% confidence level.

Western blot. Cells were collected in lysis buffer containing 25 mM HEPES, pH 7.5, 0.3 M NaCl2, 1.5 mM MgCl2, 0.2 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.5% Deoxycholic Acid, 20 mM β-glycerolphosphate plus protease and phosphatase inhibitors (0.2 mg/mL Leupeptin, 2 mg/mL Aprotinin, 1 mM PMSF and 0.1 mM Na3VO4). Protein quantification was performed using the BCA Protein Assay Kit (Pierce #23235) following the manufacturer’s instructions. 50 µg of protein were resolved on 4-15% mini protean precast gels (Bio-Rad, Hercules, CA, USA), transferred to PVDF membranes (Millipore, Darmstadt, Germany) and blotted against (Table S2): CD31 (Dako, Goat IgG, 1:1000), Von Willebrand Factor (Abcam, Mouse IgG, 1:1000), NCAM-1 (Santa-Cruz Biotech, Rabbit IgG, 1:500) and Neuropilin-2 (Santa-Cruz Biotech, Mouse monoclonal IgG, 1:500). β-Actin (Sigma-Aldrich) was used as loading control. Antigen detection was achieved by enhanced chemiluminescence (Millipore). All Western blots were imaged using a GBox (Syngene, Cambridge, United Kingdom).


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Statistical analyses. The bands of interest were analyzed for the proteomic experiments, and the proteins classified according to their quality (Matches, Covert, Mass, pl, emPAI). Unicity scores were given based on their number of occurrences in other migrations, how many migrations, and the coverage of the occurrences. Only proteins with a sufficiently high unicity score were selected. These proteins were annotated using R-project software, according to the UniProt database. For western blotting, images were quantified using Image J software (NIH), and statistical analyses were performed using a t-test. Images show one representative western blot out of three, with nearly identical results.



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Results and discussion Human induced pluripotent stem cells (hiPSc) are considered to be a new, innovative, and promising tool to produce cell models for many neurological disorders such as Charcot Marie Tooth disease (CMT)6. One limitation of these models is the difficulty in controlling the differentiation process into the cells of interest, such as motor-neurons in the case of CMT disease. The canonical technique is time consuming and costly, as it requires specific culture media and growth factors. The use of a non-invasive cell sorting method, such as SdFFF, early in the process could thus be of great interest to quickly isolate cells enriched in neural progenitors, the cellular basis of in vitro motor-neuron preparation.

HiPSc generation: The first step consisted of the controlled generation of hiPSc from DF. DF obtained from the skin biopsy of a healthy person were reprogrammed into hiPSc using episomal plasmids bearing Oct4, Sox2, Klf4, and l-Myc. Fifteen days after nucleofection, the DF formed colonies with a typical morphology and were selected for further expansion. After six months of amplification, all colonies (Figure S1A) were positive for alkaline phosphatase (Figure S1D), were able to form EB (Figure S1B), and differentiate into cell types of all three embryonic germ layers following a spontaneous differentiation protocol (Figure S1E to H). These cells also expressed pluripotency markers including Nanog, Oct3/4, and Sox2 (Figure S1J to Q), and had normal karyotypes (Figure S1C). The reprogrammed dermal fibroblasts had thus acquired the properties of human embryonic stem cells, i.e. self-renewal, pluripotency, and the ability to spontaneously generate the three embryonic germ layers: ectoderm, endoderm, and mesoderm; and could therefore be classified as hiPSc.


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SdFFF elution of hiPSc-Derived Progenitors : SdFFF is a method providing gentle, noninvasive, and label free cell sorting while maintaining cellular integrity, functionality, and sterility15. The major advantages of this method are the limitation of harmful cell interactions by using an empty channel (no stationary phase), the hyperlayer elution mode, and the absence of the need for antibody-mediated cell sorting. This last point is very important as cell markers may not be well defined (in particular for stem cells), commercial labels may not be available, and labeling could interfere with further use of the cells (culture, transplantation) or even induce differentiation (stem cells). The cell separation mechanism, described as the "hyperlayer" elution mode15,

17, 20

, is based solely on intrinsic biophysical properties of the

cells under study, which are size, density, shape, and rigidity; it is a safe cell sorting method because it greatly limits cell-surface interactions and shear forces15,

16, 17, 20

. Under the

hyperlayer mode, subpopulations of cells of the same size, density, etc., are focused in thin layers away from the accumulation wall, corresponding to their equilibrium position where the external field is exactly balanced by hydrodynamic forces. The, large and less dense cells are focused in the faster streamlines and are eluted first as the elution order of "hyperlayer" is size/density dependent17, 18, 19, 20, 21, 22, 23, 24. The experimental retention ratio Robs = void time versus retention time = t0/tR (measured by the first moment method)32 was used to determine the various average velocities, retention orders, and finally, the elution mode, as previously described15. Representative fractograms and fraction collections of hiPSc are shown in Figure 2. The first fractogram peak corresponds to unretained species (void volume peak, Robs ≈ 1) and the second to the cell population with Robs = 0.258 ± 0.008. The "hyperlayer" mode was demonstrated by the means of the field and flow rate dependence of Robs, as previously described17, 18, 19, 20, 21, 22, 23, 24. Robs for hiPSc varied from 0.236 ± 0.006 (50 g and 0.4 mL/min) to 0.316 ± 0.005 (30 g and 1.0 mL/min). In the "hyperlayer" elution mode, the samples are



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lifted away from the accumulation wall, limiting harmful cell surface interactions. By using the following equation18: s=

R obs × ω 6

1

in which ω is the channel thickness (125 µm), we calculated the value of s, the average distance from the center of the cell to the channel wall18. In the "hyperlayer" mode, s should be greater than the particle radius r, calculated from the mean cell diameter. Here, the s value was 5.4 µm for a mean cell radius measured at 5.2 µm, which is close, as was previously observed for other cell lines33. This could be explained by the elution of a complex population representing a broad dispersion, either from a phenotypical or biophysical point of view, requiring elution conditions giving a broader peak. In the "hyperlayer" mode, the elution order should be size and density dependent17, 18, 19, 20, 21, 22, 23, 24 with a decrease in cell diameter as the elution profile progresses. Here, the mean diameter varied from 13.45 µm ± 0.26 µm for cells eluted in F1 to 9.67 ± 0.03 µm for cells eluted in F3 (Coulter Counter). Our results demonstrate that hiPSc cells were eluted under the "Hyperlayer" mode as we have described for various cell species15. Our main objective was to determine whether the selected conditions and collection times resulted in sorted sub-populations with different biological properties, leading to specific lineages (hiPSc-Derived Progenitors). The hiPSc population, cultured without inducing differentiation (basic medium, Figure 1), lead to a complex mixture of stem cells and progenitors at various stages of differentiation, corresponding to different layers: ectoderm, mesoderm, and endoderm, including theEB from which they derived. This very complex population could be described as a continuum of differentiation from a biological point of view, and as a matrix of polydispersity in terms of size, density, shape, and rigidity from a biophysical point of view. It was difficult to find standards which represent the entire complexity of this matrix to characterize this population, in particular standards able to reproduce the complex variation of rigidity linked to the 14 ACS Paragon Plus Environment

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differentiation process. Biological characterization of our sub-populations was carried out using proteomic analyses. Three fractions were collected and named F1, F2, and F3 (Figure 2A). F1 contained a few cells and mostly cell debris, whereas F2 and F3 were cell-rich. We therefore focused our attention on fractions F2 and F3. The morphology of the cells obtained from F2 and F3 was very different after seven days of culture in basic medium without specific factors, which permitted lineage differentiation. SdFFF elution requires a suspension of isolated spherical cells. After a few hours in culture, the cells adhere to the support and change their morphology. Daily microscopic observation showed the evolving morphological changes depending on the cell phenotype. F2-derived cells displayed an epithelial morphology. They grew in stratified layers without extracellular spaces (cluster) (Figure 2B). F3-derived cells did not exhibit the same characteristics (Figure 2B). The nucleus to cytoplasm ratio was lower and the cells displayed long, thin, bipolar extensions (processes), typical of neural progenitors. These observations demonstrate both the nature and homogeneity of the fractions. We performed proteomic analysis to characterize these two subpopulations.



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involved in lineage differentiation. F2-derived cells

grew

in

a

stratified

layer

without

extracellular spaces. F3-derived cells had a reduced nucleus/cytoplasm ratio and displayed long bipolar extensions (white arrow) (phase contrast microcopy, scale bar = 20 µm).

Figure 2: Representative fractograms of hiPSc derived progenitors and fraction collection

(A) Representative fractograms of hiPSc derived progenitors and fraction collection. Elution conditions: flow injection of 100 µL cell suspension (2.5 × 106 cells/mL), flow rate: 0.6 mL/min (sterile PBS, pH 7.4); external multigravitational

field:

40.00

±

0.02

g,

spectrophotometric detection at λ = 254 nm. ER corresponds to the end of channel rotation (mean externally field strength = 0 g). (B) hiPSc derived progenitors after culture in basic culture medium without specific factors


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Protein analyses. One of our objectives was to produce a sub-population enriched in neural progenitors, which would represent the cellular basis for motor neuron (MN) preparation. Obtaining un-tagged sub-populations was important to preserve cell function, especially for further use of the stem cells. Moreover, specific cell markers are not clearly identified for a majority of stem cells, whereas the sub-populations could be characterized by proteomic analyses. After protein extraction and SDS-PAGE separation (Figure S2), six bands which differed in intensity between the two fractions were identified and analyzed by mass spectroscopy. They corresponded to a total of 321 different proteins (Tables SI). Among these, 158 proteins were found both in F2 and F3 at relatively similar levels, whereas 14 were more expressed in F2 than F3 and 15 were more expressed in F3 than F2. However, the most important result was that 38 and 96 proteins were only found in F2 or F3, respectively (Figure 3A). We used the UniProt database to classify the identified proteins. As expected, we found that many of the proteins present in both fractions were involved in basic cell functions, such as metabolism, extracellular matrix or apoptosis. Most of F2-specific proteins were involved in endothelial cell biology (green) (Figure 3C) whereas a few seemed to be involved in neural fate (red) (Figure 3B). In contrast, a large proportion of F3-specific proteins were involved in neural cell biological processes and a few seemed to be involved in endothelial fate (Figure 3D). These results, together with the microscopic observations, clearly show that the cells eluted in these two fractions were different. The F2 population appeared to be engaged in endothelial differentiation whereas the F3 population was engaged in neural differentiation.



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A

B

C

Figure 3: Repartition of 321 proteins only in F2, F3, or in both F2 and F3 after statistical analyses (A), unique F2 proteins (B),

or

unique

F3

proteins

(C).

Classification was performed according to biological functions given by Uniprot Data Base.


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Western blots were performed using characteristic markers of each lineage to confirm these observations (Figure 4A). We used common validated markers for each lineage because validated antibodies against F2- or F3-specific proteins were not commercially available for the vast majority of these proteins. CD-31 is also known as platelet endothelial cell adhesion molecule 1 (PECAM-1), and is a transmembrane glycoprotein. It is present on the surface of monocytes, granulocytes, platelets, and endothelial cells34. Von Willebrand’s factor (VWF) is a blood glycoprotein involved in the maintenance of hemostasis. The VWF antibody is specific for endothelial cell cytoplasm35. Neuropilin-2 (NRP-2) is a transmembrane glycoprotein receptor for semaphorins involved in axonal repulsive activity that prevents improper innervation during embryogenesis36. Roles of NRP-2 are more specific to the neural system. Neural cell adhesion molecule 1 (NCAM-1, CD56) is a homophilic binding glycoprotein specifically expressed on neuron membranes37,

38, 39

. CD31 and VWF protein

levels were significantly higher in F2, (1.65 ± 0.16 and 1.83 ± 0.49), than in F3 (0.81 ± 0.13 and 0.8 ± 0.35. p = 0.001 and p = 0.009, respectively). CD31 and VWF are specific to endothelial cells. As expected, the expression level of NRP-2 was higher in F3 than in F2 (1.36 ± 0.16 and 0.97 ± 0.24, p = 0.036). The expression level of NCAM-1 was significantly higher in F2 and F3 than in the TP fraction (p = 0.003 and p = 0.009, respectively), although there was no significant difference in the level of NCAM-1 between F2 and F3 (Figure 4B). NRP-2 and NCAM-1 are specific to neural cells. Proteomic analysis thus demonstrates that endothelial progenitor proteins were more expressed in F2 than in F3, and neural progenitor proteins were expressed in F3 (especially NRP-2).



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Figure 4: the expression level of CD-31, VWF, NRP-2, and NCAM proteins was analyzed

by

quantification

Western of

three

blot

(B):

independent

experiments performed in A. Results are presented as the Mean ± S.E.M. For statistics,

a

t-test

was

performed.

Significant differences are indicated as **p

New Method for Sorting Endothelial and Neural Progenitors from

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