Induced Pluripotent Stem Cells: Generation, Characterization, and Differentiation—Methods and Protocols

Graversen, Veronica Kon; Chavala, Sai H.

doi:10.1007/7651_2014_148

Cited by 2 publications

(2 citation statements)

References 10 publications

(12 reference statements)

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“… 8 iPSC generation using conventional monolayer cultures typically takes 6–8 weeks, with varying degrees of efficiency depending on the method of reprogramming. 5 , 9 , 10 In order to increase the reprogramming efficiency and ultimately scale up the production of these cells, researchers have tried to use bioreactor suspension culture to induce pluripotency of mouse fibroblasts to mouse iPSCs in the form of cell aggregates. 11 , 12 , 13 However, it is not yet clear whether the suspension culture approach will work well for human cells.…”

Section: Introductionmentioning

confidence: 99%

“…Importantly, the limited number of derived cells may be unable to support potential clinical applications 8 . iPSC generation using conventional monolayer cultures typically takes 6–8 weeks, with varying degrees of efficiency depending on the method of reprogramming 5,9,10 . In order to increase the reprogramming efficiency and ultimately scale up the production of these cells, researchers have tried to use bioreactor suspension culture to induce pluripotency of mouse fibroblasts to mouse iPSCs in the form of cell aggregates 11–13 .…”

Section: Introductionmentioning

confidence: 99%

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An allied reprogramming, selection, expansion and differentiation platform for creating hiPSC on microcarriers

Lam

Vassilev

et al. 2022

Cell Proliferation

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Objectives Induced pluripotent stem cells (iPSCs) generated by monolayer cultures is plagued by low efficiencies, high levels of manipulation and operator unpredictability. We have developed a platform, reprogramming, expansion, and differentiation on Microcarriers, to solve these challenges. Materials and Methods Five sources of human somatic cells were reprogrammed, selected, expanded and differentiated in microcarriers suspension cultures. Results Improvement of transduction efficiencies up to 2 times was observed. Accelerated reprogramming in microcarrier cultures was 7 days faster than monolayer, providing between 30 and 50‐fold more clones to choose from fibroblasts, peripheral blood mononuclear cells, T cells and CD34+ stem cells. This was observed to be due to an earlier induction of genes (β‐catenin, E‐cadherin and EpCAM) on day 4 versus monolayer cultures which occurred on days 14 or later. Following that, faster induction and earlier stabilization of pluripotency genes occurred during the maturation phase of reprogramming. Integrated expansion without trypsinization and efficient differentiation, without embryoid bodies formation, to the three germ‐layers, cardiomyocytes and haematopoietic stem cells were further demonstrated. Conclusions Our method can solve the inherent problems of conventional monolayer cultures. It is highly efficient, cell dissociation free, can be operated with lower labor, and allows testing of differentiation efficiency without trypsinization and generation of embryoid bodies. It is also amenable to automation for processing more samples in a small footprint, alleviating many challenges of manual monolayer selection.

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