scholarly journals Standardized quality control workflow to evaluate the reproducibility and differentiation potential of human iPSCs into neurons

2021 ◽  
Author(s):  
Carol X.-Q. Chen ◽  
Narges Abdian ◽  
Gilles Maussion ◽  
Rhalena A. Thomas ◽  
Iveta Demirova ◽  
...  
Keyword(s):  
Stem Cells ◽  
Quality Control ◽  
Cortical Neurons ◽  
Genomic Stability ◽  
Control Measures ◽  
List Type ◽  
Ipsc Line ◽  
Differentiation Potential ◽  
Germ Layers ◽  
Human Ipscs

AbstractInduced pluripotent stem cells (iPSCs) derived from human somatic cells have created new opportunities to generate disease-relevant cells. Thus, as the use of patient-derived stem cells has become more widespread, having a workflow to monitor each line is critical. This ensures iPSCs pass a suite of quality control measures, promoting reproducibility across experiments and between labs. With this in mind, we established a four-step workflow to assess our newly generated iPSCs for variations and reproducibility relative to each other and iPSCs obtained from external sources. Our benchmarks for evaluating iPSCs include examining iPSC morphology and proliferation in two different media conditions (mTeSR1 and Essential 8) and evaluating their ability to differentiate into each of the three germ layers, with a particular focus on neurons. Genomic stability in the human iPSCs was analyzed by G-band karyotyping and a qPCR-based stability test, and cell-line identity authenticated by Short Tandem Repeat (STR) analysis. Using standardized dual SMAD inhibition methods, all iPSC lines gave rise to neural progenitors that could subsequently be differentiated into cortical neurons. Neural differentiation was analyzed qualitatively by immunocytochemistry and quantitatively by q-PCR for progenitor, neuronal, cortical and glial markers. Taken together, we present a standardized quality control workflow to evaluate variability and reproducibility across and between iPSCs.HighlightsValidation of culture conditions is critical in the expansion and maintenance of an iPSC line.Characterization of pluripotency and genomic stability ensures each line is free of defects at the DNA level, while maintaining its ability to be directed into any of the three germ layers.Forebrain cortical neurons can be generated from all iPSC line tested; however, the morphology and expression pattern of these neurons can vary from line to line.

scholarly journals A Multistep Workflow to Evaluate Newly Generated iPSCs and Their Ability to Generate Different Cell Types

2021 ◽  
Vol 4 (3) ◽  
pp. 50
Author(s):  
Carol X.-Q. Chen ◽  
Narges Abdian ◽  
Gilles Maussion ◽  
Rhalena A. Thomas ◽  
Iveta Demirova ◽  
...  
Keyword(s):  
Stem Cells ◽  
Quality Control ◽  
Cell Growth ◽  
Cortical Neurons ◽  
Cell Types ◽  
Control Measures ◽  
Growth Media ◽  
Cell Type ◽  
Human Ipscs ◽  
Induced Pluripotent

Induced pluripotent stem cells (iPSCs) derived from human somatic cells have created new opportunities to generate disease-relevant cells. Thus, as the use of patient-derived stem cells has become more widespread, having a workflow to monitor each line is critical. This ensures iPSCs pass a suite of quality-control measures, promoting reproducibility across experiments and between labs. With this in mind, we established a multistep workflow to assess our newly generated iPSCs. Our workflow tests four benchmarks: cell growth, genomic stability, pluripotency, and the ability to form the three germline layers. We also outline a simple test for assessing cell growth and highlight the need to compare different growth media. Genomic integrity in the human iPSCs is analyzed by G-band karyotyping and a qPCR-based test for the detection of common karyotypic abnormalities. Finally, we confirm that the iPSC lines can differentiate into a given cell type, using a trilineage assay, and later confirm that each iPSC can be differentiated into one cell type of interest, with a focus on the generation of cortical neurons. Taken together, we present a multistep quality-control workflow to evaluate newly generated iPSCs and detail the findings on these lines as they are tested within the workflow.

scholarly journals A Src inhibitor regulates the cell cycle of human pluripotent stem cells and improves directed differentiation

2015 ◽  
Vol 210 (7) ◽  
pp. 1257-1268 ◽  
Author(s):  
Sundari Chetty ◽  
Elise N. Engquist ◽  
Elie Mehanna ◽  
Kathy O. Lui ◽  
Alexander M. Tsankov ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Human Pluripotent Stem Cells ◽  
Directed Differentiation ◽  
Differentiation Potential ◽  
Germ Layers ◽  
Positive Effects ◽  
Src Inhibitor ◽  
Expression Of Genes

Driving human pluripotent stem cells (hPSCs) into specific lineages is an inefficient and challenging process. We show that a potent Src inhibitor, PP1, regulates expression of genes involved in the G1 to S phase transition of the cell cycle, activates proteins in the retinoblastoma family, and subsequently increases the differentiation propensities of hPSCs into all three germ layers. We further demonstrate that genetic suppression of Src regulates the activity of the retinoblastoma protein and enhances the differentiation potential of hPSCs across all germ layers. These positive effects extend beyond the initial germ layer specification and enable efficient differentiation at subsequent stages of differentiation.

scholarly journals Long-Term Stability and Differentiation Potential of Cryopreserved cGMP-Compliant Human Induced Pluripotent Stem Cells

2019 ◽  
Vol 21 (1) ◽  
pp. 108 ◽  
Author(s):  
Mehdi Shafa ◽  
Tylor Walsh ◽  
Krishna Morgan Panchalingam ◽  
Thomas Richardson ◽  
Laura Menendez ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Genomic Stability ◽  
Directed Differentiation ◽  
Differentiation Potential ◽  
Long Term Stability ◽  
Induced Pluripotent ◽  
Cell Banks ◽  
2D And 3D

The clinical effectiveness of human induced pluripotent stem cells (iPSCs) is highly dependent on a few key quality characteristics including the generation of high quality cell bank, long-term genomic stability, post-thaw viability, plating efficiency, retention of pluripotency, directed differentiation, purity, potency, and sterility. We have already reported the establishment of iPSC master cell banks (MCBs) and working cell banks (WCBs) under current good manufacturing procedure (cGMP)-compliant conditions. In this study, we assessed the cellular and genomic stability of the iPSC lines generated and cryopreserved five years ago under cGMP-compliant conditions. iPSC lines were thawed, characterized, and directly differentiated into cells from three germ layers including cardiomyocytes (CMs), neural stem cells (NSCs), and definitive endoderm (DE). The cells were also expanded in 2D and 3D spinner flasks to evaluate their long-term expansion potential in matrix-dependent and feeder-free culture environment. All three lines successfully thawed and attached to the L7TM matrix, and formed typical iPSC colonies that expressed pluripotency markers over 15 passages. iPSCs maintained their differentiation potential as demonstrated with spontaneous and directed differentiation to the three germ layers and corresponding expression of specific markers, respectfully. Furthermore, post-thaw cells showed normal karyotype, negative mycoplasma, and sterility testing. These cells maintained both their 2D and 3D proliferation potential after five years of cryopreservation without acquiring karyotype abnormality, loss of pluripotency, and telomerase activity. These results illustrate the long-term stability of cGMP iPSC lines, which is an important step in establishing a reliable, long-term source of starting materials for clinical and commercial manufacturing of iPSC-derived cell therapy products.

scholarly journals Using induced pluripotent stem cells to investigate human neuronal phenotypes in 1q21.1 deletion and duplication syndrome

2021 ◽  
Author(s):  
Gareth Chapman ◽  
Mouhamed Alsaqati ◽  
Sharna Lunn ◽  
Tanya Singh ◽  
Stefanie C Linden ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Cortical Neurons ◽  
Neuronal Development ◽  
Motor Deficits ◽  
Differentiation Potential ◽  
Induced Pluripotent ◽  
The Impact ◽  
1Q21.1 Deletion

AbstractCopy Number Variation (CNV) at the 1q21.1 locus is associated with a range of neurodevelopmental and psychiatric disorders in humans, including abnormalities in head size and motor deficits. Yet, the functional consequences of these CNVs (both deletion and duplication) on neuronal development remain unknown. To determine the impact of CNV at the 1q21.1 locus on neuronal development, we generated induced pluripotent stem cells from individuals harbouring 1q21.1 deletion or duplication and differentiated them into functional cortical neurons. We show that neurons with 1q21.1 deletion or duplication display reciprocal phenotype with respect to proliferation, differentiation potential, neuronal maturation, synaptic density, and functional activity. Deletion of the 1q21.1 locus was also associated with an increased expression of lower cortical layer markers. This difference was conserved in the mouse model of 1q21.1 deletion, which displayed altered corticogenesis. Importantly, we show that neurons with 1q21.1 deletion and duplication are associated with differential expression of calcium channels and demonstrate that physiological deficits in neurons with 1q21.1 deletion or duplication can be pharmacologically modulated by targeting Ca2+ channel activity. These findings provide biological insight into the neuropathological mechanism underlying 1q21.1 associated brain disorder and indicate a potential target for therapeutic interventions.

scholarly journals Generation of cortical neurons through large-scale expanding neuroepithelial stem cell from human pluripotent stem cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Shumei Zhao ◽  
Kui Duan ◽  
Zongyong Ai ◽  
Baohua Niu ◽  
Yanying Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Suspension Culture ◽  
Pluripotent Stem Cells ◽  
Cortical Neurons ◽  
Culture System ◽  
Large Scale ◽  
Disease Modeling ◽  
High Quality ◽  
Differentiation Potential

Abstract Background Considerable progress has been made in converting human pluripotent stem cells (hPSCs) into cortical neurons for disease modeling and regenerative medicine. However, these procedures are hard to provide sufficient cells for their applications. Using a combination of small-molecules and growth factors, we previously identified one condition which can rapidly induce hPSCs into neuroepithelial stem cells (NESCs). Here, we developed a scalable suspension culture system, which largely yields high-quality NESC-spheres and subsequent cortical neurons. Methods The NESC medium was first optimized, and the suspension culture system was then enlarged from plates to stirred bioreactors for large-scale production of NESC-spheres by a stirring speed of 60 rpm. During the expansion, the quality of NESC-spheres was evaluated. The differentiation potential of NESC-spheres into cortical neurons was demonstrated by removing bFGF and two pathway inhibitors from the NESC medium. Cellular immunofluorescence staining, global transcriptome, and single-cell RNA sequencing analysis were used to identify the characteristics, identities, purities, or homogeneities of NESC-spheres or their differentiated cells, respectively. Results The optimized culture system is more conducive to large-scale suspension production of NESCs. These largely expanded NESC-spheres maintain unlimited self-renewal ability and NESC state by retaining their uniform sizes, high cell vitalities, and robust expansion abilities. After long-term expansion, NESC-spheres preserve high purity, homogeneity, and normal diploid karyotype. These expanded NESC-spheres on a large scale have strong differentiation potential and effectively produce mature cortical neurons. Conclusions We developed a serum-free, defined, and low-cost culture system for large-scale expansion of NESCs in stirred suspension bioreactors. The stable and controllable 3D system supports long-term expansion of high-quality and homogeneous NESC-spheres. These NESC-spheres can be used to efficiently give rise to cortical neurons for cell therapy, disease modeling, and drug screening in future.

scholarly journals Targeted gene correction of FKRP by CRISPR/Cas9 restores functional glycosylation of α-dystroglycan in cortical neurons derived from human induced pluripotent stem cells

2017 ◽  
Author(s):  
Beatrice Lana ◽  
Jihee Kim ◽  
David Ryan ◽  
Evangelos Konstantinidis ◽  
Sandra Louzada ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Genome Editing ◽  
Gene Mutation ◽  
Pluripotent Stem Cells ◽  
Cortical Neurons ◽  
List Type ◽  
Directed Differentiation ◽  
Gene Correction ◽  
Induced Pluripotent

SummaryMutations in genes required for functional glycosylation of α-dystroglycan cause a group of congenital muscular dystrophies associated with brain malformations, referred to as dystroglycanopathies. The lack of isogenic, physiology-relevant human cellular models has limited our understanding of the cortical abnormalities in dystroglycanopathies. Here we generate induced pluripotent stem cells (iPSCs) from a severe dystroglycanopathy patient with homozygous mutations in the ribitol-5-phosphate transferase gene, FKRP. We carry out targeted gene correction in FKRP-iPSCs using CRISPR/Cas9-mediated genome editing. We characterise the directed differentiation of FKRP- and corrected-iPSCs to neural stem cells, cortical progenitors and cortical neurons. Importantly, we show that targeted gene correction of FKRP restores functional glycosylation of α-dystroglycan in iPSC-derived cortical neurons. We independently validate this result by showing targeted gene mutation of FKRP disrupts functional glycosylation of α-dystroglycan. This work demonstrates the feasibility of using CRISPR/Cas9-engineered human iPSCs for modelling dystroglycanopathies and provides a foundation for therapeutic development.HighlightsGeneration of FKRP-iPSCs for modelling cortical abnormalities in dystroglycanopathiesPrecise gene correction by CRISPR/Cas9-mediated genome editingDirected differentiation of isogenic control and FKRP-iPSC to cortical neuronsFunctional glycosylation of α-dystroglycan is restored in cortical neurons derived from CRISPR/Cas9-corrected iPSCsTargeted gene mutation of FKRP disrupts functional glycosylation of α-dystroglycan in cortical neurons

scholarly journals Development and Application of 3D Bioprinted Scaffolds Supporting Induced Pluripotent Stem Cells

2021 ◽  
Vol 2021 ◽  
pp. 1-13
Author(s):  
Dezhi Lu ◽  
Yang Liu ◽  
Wentao Li ◽  
Hongshi Ma ◽  
Tao Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Three Dimensional ◽  
Layer By Layer ◽  
3D Bioprinting ◽  
Differentiation Potential ◽  
Human Ipscs ◽  
Induced Pluripotent

Three-dimensional (3D) bioprinting is a revolutionary technology that replicates 3D functional living tissue scaffolds in vitro by controlling the layer-by-layer deposition of biomaterials and enables highly precise positioning of cells. With the development of this technology, more advanced research on the mechanisms of tissue morphogenesis, clinical drug screening, and organ regeneration may be pursued. Because of their self-renewal characteristics and multidirectional differentiation potential, induced pluripotent stem cells (iPSCs) have outstanding advantages in stem cell research and applications. In this review, we discuss the advantages of different bioinks containing human iPSCs that are fabricated by using 3D bioprinting. In particular, we focus on the ability of these bioinks to support iPSCs and promote their proliferation and differentiation. In addition, we summarize the applications of 3D bioprinting with iPSC-containing bioinks and put forward new views on the current research status.

scholarly journals Using induced pluripotent stem cells to investigate human neuronal phenotypes in 1q21.1 deletion and duplication syndrome

2021 ◽  
Author(s):  
Gareth Chapman ◽  
Mouhamed Alsaqati ◽  
Sharna Lunn ◽  
Tanya Singh ◽  
Stefanie C. Linden ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Cortical Neurons ◽  
Neuronal Development ◽  
Motor Deficits ◽  
Differentiation Potential ◽  
Induced Pluripotent ◽  
The Impact ◽  
1Q21.1 Deletion

AbstractCopy Number Variation (CNV) at the 1q21.1 locus is associated with a range of neurodevelopmental and psychiatric disorders in humans, including abnormalities in head size and motor deficits. Yet, the functional consequences of these CNVs (both deletion and duplication) on neuronal development remain unknown. To determine the impact of CNV at the 1q21.1 locus on neuronal development, we generated induced pluripotent stem cells from individuals harbouring 1q21.1 deletion or duplication and differentiated them into functional cortical neurons. We show that neurons with 1q21.1 deletion or duplication display reciprocal phenotype with respect to proliferation, differentiation potential, neuronal maturation, synaptic density and functional activity. Deletion of the 1q21.1 locus was also associated with an increased expression of lower cortical layer markers. This difference was conserved in the mouse model of 1q21.1 deletion, which displayed altered corticogenesis. Importantly, we show that neurons with 1q21.1 deletion and duplication are associated with differential expression of calcium channels and demonstrate that physiological deficits in neurons with 1q21.1 deletion or duplication can be pharmacologically modulated by targeting Ca2+ channel activity. These findings provide biological insight into the neuropathological mechanism underlying 1q21.1 associated brain disorder and indicate a potential target for therapeutic interventions.

scholarly journals A New Predictive Technology for Perinatal Stem Cell Isolation Suited for Cell Therapy Approaches

Micromachines ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 782
Author(s):  
Silvia Zia ◽  
Giulia Martini ◽  
Valeria Pizzuti ◽  
Alessia Maggio ◽  
Giuliana Simonazzi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Quality Control ◽  
Stem Cell ◽  
New Technology ◽  
Cell Isolation ◽  
Individual Variability ◽  
Label Free ◽  
Differentiation Potential ◽  
Proliferation Ability ◽  
Stem Cell Isolation

The use of stem cells for regenerative applications and immunomodulatory effect is increasing. Amniotic epithelial cells (AECs) possess embryonic-like proliferation ability and multipotent differentiation potential. Despite the simple isolation procedure, inter-individual variability and different isolation steps can cause differences in isolation yield and cell proliferation ability, compromising reproducibility observations among centers and further applications. We investigated the use of a new technology as a diagnostic tool for quality control on stem cell isolation. The instrument label-free separates cells based on their physical characteristics and, thanks to a micro-camera, generates a live fractogram, the fingerprint of the sample. Eight amniotic membranes were processed by trypsin enzymatic treatment and immediately analysed. Two types of profile were generated: a monomodal and a bimodal curve. The first one represented the unsuccessful isolation with all recovered cell not attaching to the plate; while for the second type, the isolation process was successful, but we discovered that only cells in the second peak were alive and resulted adherent. We optimized a Quality Control (QC) method to define the success of AEC isolation using the fractogram generated. This predictive outcome is an interesting tool for laboratories and cell banks that isolate and cryopreserve fetal annex stem cells for research and future clinical applications.

scholarly journals Endowing iPSC-Derived MSCs with Angiogenic and Keratinogenic Differentiation Potential: A Promising Cell Source for Skin Tissue Engineering

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Weimin Lin ◽  
Miao Chen ◽  
Chen Hu ◽  
Siyu Qin ◽  
Chenyu Chu ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Growth Factor ◽  
Cell Line ◽  
Skin Tissue ◽  
Differentiation Potential ◽  
Skin Tissue Engineering ◽  
Cell Based Therapy ◽  
Human Ipscs ◽  
Cell Source

Induced pluripotent stem cells (iPSC) hold tremendous potential for personalized cell-based therapy for skin regeneration. Aiming to establish human iPSCs as a potential cell source for skin tissue engineering, we expect to obtain an epidermal-like cell line with angiogenic and keratinogenic differentiation potential via inducing iPSC-derived mesenchymal stem cells (iPSC-MSCs) with basic fibroblast growth factor (bFGF) and/or keratinocyte growth factor (KGF). The results show that iPSC-MSCs were successfully induced with a positive FGFR/KGFR expression on the cell surface. BFGF/KGF induction could significantly increase the expression of vascularization marker CD31 and keratinization marker CK10, respectively, while when combined together, although CD31 and CK10 were still positively expressed, their expressions were lower than that of the single induction group, suggesting that the effects of the two growth factors interfered with each other. This cell line with angiogenic and keratinogenic differentiation potential provides a promising new source of cells for the construction of well vascularized and keratinized tissue engineered skin, furthermore establishing an effective strategy for iPSC-based therapy in skin tissue engineering.

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