To Clone or Not to Clone? Induced Pluripotent Stem Cells Can Be Generated in Bulk Culture

Willmann, Charlotte A.; Hemeda, Hatim; Pieper, Lisa A.; Lenz, Michael; Qin, Jie; Joussen, Sylvia; Sontag, Stephanie; Wanek, Paul; Denecke, Bernd; Schüler, H; Zenke, Martin; Wagner, Wolfgang

doi:10.1371/journal.pone.0065324

Cited by 37 publications

(37 citation statements)

References 50 publications

(62 reference statements)

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“…Other groups have also started to perform similar selection by pooling iPS cells derived from human fibroblasts [48,49]. Both studies showed that high-quality iPS cells can be obtained from pooling of bulk cultures and expanded more efficiently than using individual colonies.…”

Section: Discussionmentioning

confidence: 99%

A Facile Method to Establish Human Induced Pluripotent Stem Cells From Adult Blood Cells Under Feeder-Free and Xeno-Free Culture Conditions: A Clinically Compliant Approach

Chou

Gao

et al. 2015

Stem Cells Translational Medicine

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Reprogramming human adult blood mononuclear cells (MNCs) cells by transient plasmid expression is becoming increasingly popular as an attractive method for generating induced pluripotent stem (iPS) cells without the genomic alteration caused by genome-inserting vectors. However, its efficiency is relatively low with adult MNCs compared with cord blood MNCs and other fetal cells and is highly variable among different adult individuals. We report highly efficient iPS cell derivation under clinically compliant conditions via three major improvements. First, we revised a combination of three EBNA1/OriP episomal vectors expressing five transgenes, which increased reprogramming efficiency by ‡10-50-fold from our previous vectors. Second, human recombinant vitronectin proteins were used as cell culture substrates, alleviating the need for feeder cells or animal-sourced proteins. Finally, we eliminated the previously critical step of manually picking individual iPS cell clones by pooling newly emerged iPS cell colonies. Pooled cultures were then purified based on the presence of the TRA-1-60 pluripotency surface antigen, resulting in the ability to rapidly expand iPS cells for subsequent applications. These new improvements permit a consistent and reliable method to generate human iPS cells with minimal clonal variations from blood MNCs, including previously difficult samples such as those from patients with paroxysmal nocturnal hemoglobinuria. In addition, this method of efficiently generating iPS cells under feeder-free and xeno-free conditions allows for the establishment of clinically compliant iPS cell lines for future therapeutic applications. STEM CELLS

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Section: Discussionmentioning

confidence: 99%

A Facile Method to Establish Human Induced Pluripotent Stem Cells From Adult Blood Cells Under Feeder-Free and Xeno-Free Culture Conditions: A Clinically Compliant Approach

Chou

Gao

et al. 2015

Stem Cells Translational Medicine

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“…Because of the size limitation for gene transfer using lentivirus (~9–10 kb between LTRs [27]), we had to use a short version (~1.7 kb) of the 3′ end of the classical 6.5 kb-long α-MHC promoter. Although this fragment contained important gene expression regulatory elements (TATA box, MEF-1 MEF-2 and Nkx2.5 binding sites) [34], it is likely that unidentified enhancing elements were not present explaining the weak GFP expression.…”

Section: Discussionmentioning

confidence: 99%

Scalable Electrophysiological Investigation of iPS Cell-Derived Cardiomyocytes Obtained by a Lentiviral Purification Strategy

Friedrichs

Malan

Voss

et al. 2015

JCM

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Disease-specific induced pluripotent stem (iPS) cells can be generated from patients and differentiated into functional cardiomyocytes for characterization of the disease and for drug screening. In order to obtain pure cardiomyocytes for automated electrophysiological investigation, we here report a novel non-clonal purification strategy by using lentiviral gene transfer of a puromycin resistance gene under the control of a cardiac-specific promoter. We have applied this method to our previous reported wild-type and long QT syndrome 3 (LQTS 3)-specific mouse iPS cells and obtained a pure cardiomyocyte population. These cells were investigated by action potential analysis with manual and automatic planar patch clamp technologies, as well as by recording extracellular field potentials using a microelectrode array system. Action potentials and field potentials showed the characteristic prolongation at low heart rates in LQTS 3-specific, but not in wild-type iPS cell-derived cardiomyocytes. Hence, LQTS 3-specific cardiomyocytes can be purified from iPS cells with a lentiviral strategy, maintain the hallmarks of the LQTS 3 disease and can be used for automated electrophysiological characterization and drug screening.

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“…This shortening of the repeat was sufficient to restore normal levels of DNA methylation at the locus, allow FMRP expression, and rescued the neural progenitor cells in terms of neurodevelopmental deficits 14 . These observations point to the importance of repeat length in epigenetic silencing of FMRP and highlight the need to consider somatic mosaicism (or repeat instability) along with the need to characterize multiple clones from subjects if single cell clones are selected 16 . Importantly in terms of replication of findings in different FMR1 mutation lines, similar neurodevelopmental deficits have been reported in both human embryonic stem cell (hESC) models 12,13 and in other iPSC models 15 .…”

Section: Phenotypic Assays In Human Ipsc Models Of Neuropsychiatrimentioning

confidence: 97%

Advancing drug discovery for neuropsychiatric disorders using patient-specific stem cell models

Haggarty

Silva

Cross

et al. 2016

Molecular and Cellular Neuroscience

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Compelling clinical, social, and economic reasons exist to innovate in the process of drug discovery for neuropsychiatric disorders. The use of patient-specific, induced pluripotent stem cells (iPSCs) now affords the ability to generate neuronal cell-based models that recapitulate key aspects of human disease. In the context of neuropsychiatric disorders, where access to physiologically active and relevant cell types of the central nervous system for research is extremely limiting, iPSC-derived in vitro culture of human neurons and glial cells is transformative. Potential applications relevant to early stage drug discovery, include support of quantitative biochemistry, functional genomics, proteomics, and perhaps most notably, high-throughput and high-content chemical screening. While many phenotypes in human iPSC-derived culture systems may prove adaptable to screening formats, addressing the question of which in vitro phenotypes are ultimately relevant to disease pathophysiology and therefore more likely to yield effective pharmacological agents that are disease-modifying treatments requires careful consideration. Here, we review recent examples of studies of neuropsychiatric disorders using human stem cell models where cellular phenotypes linked to disease and functional assays have been reported. We also highlight technical advances using genome-editing technologies in iPSCs to support drug discovery efforts, including the interpretation of the functional significance of rare genetic variants of unknown significance and for the purpose of creating cell type- and pathway-selective functional reporter assays. Additionally, we evaluate the potential of in vitro stem cell models to investigate early events of disease pathogenesis, in an effort to understand the underlying molecular mechanism, including the basis of selective cell-type vulnerability, and the potential to create new cell-based diagnostics to aid in the classification of patients and subsequent selection for clinical trials. A number of key challenges remain, including the scaling of iPSC models to larger cohorts and integration with rich clinicopathological information and translation of phenotypes. Still, the overall use of iPSC-based human cell models with functional cellular and biochemical assays holds promise for supporting the discovery of next-generation neuropharmacological agents for the treatment and ultimately prevention of a range of severe mental illnesses.

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To Clone or Not to Clone? Induced Pluripotent Stem Cells Can Be Generated in Bulk Culture

Cited by 37 publications

References 50 publications

A Facile Method to Establish Human Induced Pluripotent Stem Cells From Adult Blood Cells Under Feeder-Free and Xeno-Free Culture Conditions: A Clinically Compliant Approach

A Facile Method to Establish Human Induced Pluripotent Stem Cells From Adult Blood Cells Under Feeder-Free and Xeno-Free Culture Conditions: A Clinically Compliant Approach

Scalable Electrophysiological Investigation of iPS Cell-Derived Cardiomyocytes Obtained by a Lentiviral Purification Strategy

Advancing drug discovery for neuropsychiatric disorders using patient-specific stem cell models

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