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

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 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.

Optimized Protocol to Generate Spinal Motor Neuron Cells from Induced Pluripotent Stem Cells from Charcot Marie Tooth Patients

“…In addition, calcitriol is involved in various processes, such as neural stem cell differentiation, axon genesis, and the growth of MNs [48]. Furthermore, the high purity of the cells obtained with this protocol could be further improved by sedimentation field flow fractionation (SdFFF), which makes it possible to obtain neural and endothelial precursors after spontaneous differentiation in basic medium, with no added factors [49].…”

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

“…Thus, larger and less dense cells generating more hydrodynamic lift forces within the mobile phase and not very sensitive to centrifugal force (blue cells on Figure 5A ), will be eluted before smaller and denser cells (red cells, Figure 5A ) generating less hydrodynamic forces and more sensitive to the multigravitational field. As for a number of other cell models [ 27 , 29 , 31 ], we found that under the elution conditions, U87 cells were eluted under the hyperlayer elution mode [ 28 , 30 ] schematized in Figure 5A .…”

“…A pioneering study of CSCs sorting from within a GB cell line [ 28 ] made it possible to sort other cancer cells [ 29 ] and to improve cell culture conditions ahead of cell sorting and CSCs detection [ 30 ]. Moreover, these studies allowed an opening towards the field of stem cell biology [ 31 ].…”

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

“…The usually described SdFFF elution mode for cells is the "hyperlayer" mode 22,23,32,33 in which subpopulations of cells are focused into a thin layer at an equilibrium position in the channel thickness, depending on their biophysical properties: size and density as first-order parameters, along with shape and rigidity. 24 Hyperlayer elution order is size-and density-dependent: larger and less dense cells are focused in the faster streamlines, and are consequently eluted first. The experimental retention ratio, R obs (void time divided by retention time [t 0 / t R ], measured by the first moment method) 34 , was calculated to determine the average velocities and elution modes.…”

“…One of the most interesting label-free cell sorting methods is the now well-known sedimentation field-flow fractionation (SdFFF) technique. [22][23][24] . The SdFFF is a gentle and noninvasive technique.…”