scholarly journals Single cell transfection of human-induced pluripotent stem cells using a droplet-based microfluidic system

2022 ◽  
Vol 9 (1) ◽  
Author(s):  
Camilo Pérez-Sosa ◽  
Anahí Sanluis-Verdes ◽  
Ariel Waisman ◽  
Antonella Lombardi ◽  
Gustavo Rosero ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Single Cell ◽  
Pluripotent Stem Cells ◽  
High Performance ◽  
Transfection Efficiency ◽  
Single Cells ◽  
Nucleotide Sequencing ◽  
Starting Point ◽  
Induced Pluripotent

Microfluidic tools have recently made possible many advances in biological and biomedical research. Research in fields such as physics, engineering, chemistry and biology have combined to produce innovation in microfluidics which has positively impacted diverse areas such as nucleotide sequencing, functional genomics, single-cell studies, single molecules assays and biomedical diagnostics. Among these areas, regenerative medicine and stem cells have benefited from microfluidics since these tools have had a profound impact on their applications. In this study, we present a high-performance droplet-based system for transfecting individual human-induced pluripotent stem cells. We will demonstrate that this system has great efficiency in single cells and captured droplets, like other microfluidic methods but with lower cost. Moreover, this microfluidic approach can be associated with the PiggyBac transposase-based system to increase its transfection efficiency. Our results provide a starting point for subsequent applications in more complex transfection systems, single-cell differentiation interactions, cell subpopulations and cell therapy, among other potential applications.

scholarly journals Single cell transfection of human induced pluripotent stem cells using a droplet-based microfluidic system

2020 ◽  
Author(s):  
C. Pérez ◽  
A. Sanluis-Verdes ◽  
A. Waisman ◽  
A. Lombardi ◽  
G. Rosero ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Single Cell ◽  
Pluripotent Stem Cells ◽  
High Performance ◽  
Transfection Efficiency ◽  
Single Cells ◽  
Microfluidic System ◽  
Starting Point ◽  
Induced Pluripotent

ABSTRACTMicrofluidic tools have recently made possible many advances in biological and biomedical research. Research fields such as Physics, Engineering, Chemistry and Biology have combined to produce innovation in Microfluidics which has positively impacted on areas as diverse as nucleotide sequence, functional genomics, single-cell studies, single molecules assays, and biomedical diagnostics. Among these areas regenerative medicine and stem cells have benefited from Microfluidics due to these tools have had a profound impact on their applications. In the study, we present a high-performance droplet-based system for transfecting individual human-induced pluripotent stem cells. We show that this system has great efficiency in single cells and captured droplets, similar to other microfluidic methods and lower cost. We demonstrate that this microfluidic approach can be associated with the PiggyBac transposase-based system to increase its transfection efficiency. Our results provide a starting point for subsequent applications in more complex transfection systems, single-cell differentiation interactions, cell subpopulations, cell therapy, among other potential applications.

scholarly journals Single-cell phenotyping of human induced pluripotent stem cells by high-throughput imaging

2015 ◽  
Author(s):  
Hans Christian Volz ◽  
Florian Heigwer ◽  
Tatjana Wuest ◽  
Marta Galach ◽  
Jochen Utikal ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Single Cell ◽  
High Throughput ◽  
Pluripotent Stem Cells ◽  
Single Cells ◽  
Phenotypic Variability ◽  
Screening Experiments ◽  
Induced Pluripotent ◽  
High Throughput Imaging

Single-cell phenotyping promises to yield insights into biological responses in heterogeneous cell populations. We developed a method based on single-cell analysis to phenotype human induced pluripotent stem cells (hIPSC) by high-throughput imaging. Our method uses markers for morphology and pluripotency as well as social features to characterize perturbations using a meta-phenotype based on mapping single cells to distinct phenotypic classes. Analysis of perturbations on a single cell level enhances the applicability of human induced pluripotent stem cells (hIPSC) for screening experiments taking the inherently increased phenotypic variability of these cells into account. We adapted miniaturized culture conditions to allow for the utilization of hIPSC in RNA interference (RNAi) high-throughput screens and single cell phenotyping by image analysis. We identified key regulators of pluripotency in hIPSC masked in a population-averaged analysis and we confirmed several candidate genes (SMG1, TAF1) and assessed their effect on pluripotency.

scholarly journals Single-cell RNA-seq of human induced pluripotent stem cells reveals cellular heterogeneity and cell state transitions between subpopulations

2017 ◽  
Author(s):  
Quan H. Nguyen ◽  
Samuel W. Lukowski ◽  
Han Sheng Chiu ◽  
Anne Senabouth ◽  
Timothy J. C. Bruxner ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Pluripotent Stem Cells ◽  
Single Cells ◽  
State Transitions ◽  
Transcriptional Level ◽  
Single Cell Rna Sequencing ◽  
Induced Pluripotent

AbstractHeterogeneity of cell states represented in pluripotent cultures have not been described at the transcriptional level. Since gene expression is highly heterogeneous between cells, single-cell RNA sequencing can be used to identify how individual pluripotent cells function. Here, we present results from the analysis of single-cell RNA sequencing data from 18,787 individual WTC CRISPRi human induced pluripotent stem cells. We developed an unsupervised clustering method, and through this identified four subpopulations distinguishable on the basis of their pluripotent state including: a core pluripotent population (48.3%), proliferative (47.8%), early-primed for differentiation (2.8%) and late-primed for differentiation (1.1%). For each subpopulation we were able to identify the genes and pathways that define differences in pluripotent cell states. Our method identified four transcriptionally distinct predictor gene sets comprised of 165 unique genes that denote the specific pluripotency states; and using these sets, we developed a multigenic machine learning prediction method to accurately classify single cells into each of the subpopulations. Compared against a set of established pluripotency markers, our method increases prediction accuracy by 10%, specificity by 20%, and explains a substantially larger proportion of deviance (up to 3-fold) from the prediction model. Finally, we developed an innovative method to predict cells transitioning between subpopulations, and support our conclusions with results from two orthogonal pseudotime trajectory methods.

scholarly journals Single cell transcriptional profiling reveals heterogeneity of human induced pluripotent stem cells

2011 ◽  
Vol 121 (3) ◽  
pp. 1217-1221 ◽  
Author(s):  
Kazim H. Narsinh ◽  
Ning Sun ◽  
Veronica Sanchez-Freire ◽  
Andrew S. Lee ◽  
Patricia Almeida ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Single Cell ◽  
Pluripotent Stem Cells ◽  
Transcriptional Profiling ◽  
Induced Pluripotent

scholarly journals Non-viral vector based gene transfection with human induced pluripotent stem cells derived cardiomyocytes

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Shihua Tan ◽  
Zhonghao Tao ◽  
Szejie Loo ◽  
Liping Su ◽  
Xin Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Viability ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Viral Vector ◽  
Cell Model ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Green Fluorescence Protein ◽  
Induced Pluripotent

Abstract Non-viral transfection of mammalian cardiomyocytes (CMs) is challenging. The current study aims to characterize and determine the non-viral vector based gene transfection efficiency with human induced pluripotent stem cells (hiPSCs) derived cardiomyocytes (hiPSC-CMs). hiPSC-CMs differentiated from PCBC hiPSCs were used as a cell model to be transfected with plasmids carrying green fluorescence protein (pGFP) using polyethylenimine (PEI), including Transporter 5 Transfection Reagent (TR5) and PEI25, and liposome, including lipofectamine-2000 (Lipo2K), lipofectamine-3000 (Lipo3K), and Lipofectamine STEM (LipoSTEM). The gene transfection efficiency and cell viability were quantified by flow cytometry. We found that the highest gene transfection efficiency in hiPSC-CMs on day 14 of contraction can be achieved by LipoSTEM which was about 32.5 ± 6.7%. However, it also cuased poor cell viability (60.1 ± 4.5%). Furthermore, a prolonged culture of (transfection on day 23 of contraction) hiPSC-CMs not only improved gene transfection (54.5 ± 8.9%), but also enhanced cell viability (74 ± 4.9%) by LipoSTEM. Based on this optimized gene transfection condition, the highest gene transfection efficiency was 55.6 ± 7.8% or 34.1 ± 4%, respectively, for P1C1 or DP3 hiPSC line that was derived from healthy donor (P1C1) or patient with diabetes (DP3). The cell viability was 80.8 ± 5.2% or 92.9 ± 2.24%, respectively, for P1C1 or DP3. LipoSTEM is a better non-viral vector for gene transfection of hiPSC-CMs. The highest pGFP gene transfection efficiency can reach >50% for normal hiPSC-CMs or >30% for diabetic hiPSC-CMs.

Characterization of the single-cell derived bovine induced pluripotent stem cells

2017 ◽  
Vol 49 (5) ◽  
pp. 521-527 ◽  
Author(s):  
Lixia Zhao ◽  
Zixin Wang ◽  
Jindun Zhang ◽  
Jian Yang ◽  
Xuefei Gao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Single Cell ◽  
Pluripotent Stem Cells ◽  
Induced Pluripotent

Single-cell cloning and expansion of human induced pluripotent stem cells by a microfluidic culture device

2014 ◽  
Vol 453 (1) ◽  
pp. 131-137 ◽  
Author(s):  
Taku Matsumura ◽  
Kazuya Tatsumi ◽  
Yuichiro Noda ◽  
Naoyuki Nakanishi ◽  
Atsuhito Okonogi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Single Cell ◽  
Pluripotent Stem Cells ◽  
Cell Cloning ◽  
Single Cell Cloning ◽  
Induced Pluripotent
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