scholarly journals Large-Scale Analysis of Loss of Imprinting in Human Pluripotent Stem Cells

Cell Reports ◽  
2017 ◽  
Vol 19 (5) ◽  
pp. 957-968 ◽  
Author(s):  
Shiran Bar ◽  
Maya Schachter ◽  
Talia Eldar-Geva ◽  
Nissim Benvenisty
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Large Scale ◽  
Human Pluripotent Stem Cells ◽  
Scale Analysis ◽  
Loss Of Imprinting ◽  
Large Scale Analysis

scholarly journals Modulating cell state to enhance suspension expansion of human pluripotent stem cells

2018 ◽  
Vol 115 (25) ◽  
pp. 6369-6374 ◽  
Author(s):  
Yonatan Y. Lipsitz ◽  
Curtis Woodford ◽  
Ting Yin ◽  
Jacob H. Hanna ◽  
Peter W. Zandstra
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Large Scale ◽  
Cell Types ◽  
Human Pluripotent Stem Cells ◽  
The Body ◽  
Altered Expression ◽  
Pancreatic Progenitors ◽  
Erk Inhibition

The development of cell-based therapies to replace missing or damaged tissues within the body or generate cells with a unique biological activity requires a reliable and accessible source of cells. Human pluripotent stem cells (hPSC) have emerged as a strong candidate cell source capable of extended propagation in vitro and differentiation to clinically relevant cell types. However, the application of hPSC in cell-based therapies requires overcoming yield limitations in large-scale hPSC manufacturing. We explored methods to convert hPSC to alternative states of pluripotency with advantageous bioprocessing properties, identifying a suspension-based small-molecule and cytokine combination that supports increased single-cell survival efficiency, faster growth rates, higher densities, and greater expansion than control hPSC cultures. ERK inhibition was found to be essential for conversion to this altered state, but once converted, ERK inhibition led to a loss of pluripotent phenotype in suspension. The resulting suspension medium formulation enabled hPSC suspension yields 5.7 ± 0.2-fold greater than conventional hPSC in 6 d, for at least five passages. Treated cells remained pluripotent, karyotypically normal, and capable of differentiating into all germ layers. Treated cells could also be integrated into directed differentiated strategies as demonstrated by the generation of pancreatic progenitors (NKX6.1+/PDX1+ cells). Enhanced suspension-yield hPSC displayed higher oxidative metabolism and altered expression of adhesion-related genes. The enhanced bioprocess properties of this alternative pluripotent state provide a strategy to overcome cell manufacturing limitations of hPSC.

scholarly journals Development of a modular automated system for maintenance and differentiation of adherent human pluripotent stem cells

2016 ◽  
Author(s):  
Duncan E. Crombie ◽  
Maciej Daniszewski ◽  
Helena H. Liang ◽  
Tejal Kulkarni ◽  
Fan Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Large Scale ◽  
Human Pluripotent Stem Cells ◽  
Automated System ◽  
Patient Specific ◽  
High Throughput Analysis ◽  
Large Scale Culture ◽  
Induced Pluripotent ◽  
Selection Of

AbstractPatient-specific induced pluripotent stem cells (iPSCs) have tremendous potential for development of regenerative medicine, disease modelling and drug discovery. However, the processes of reprogramming, maintenance and differentiation are labour intensive and subject to inter-technician variability. To address these issues, we established and optimised protocols to allow for the automated maintenance of reprogrammed somatic cells into iPSCs to enable the large-scale culture and passaging of human pluripotent stem cells (PSCs) using a customized TECAN Freedom EVO. Generation of iPSCs was performed offline by nucleofection followed by selection of TRA-1-60 positive cells using a Miltenyi MultiMACS24 Separator. Pluripotency markers were assessed to confirm pluripotency of the generated iPSCs. Passaging was performed using an enzyme-free dissociation method. Proof of concept of differentiation was obtained by differentiating human PSCs into cells of the retinal lineage. Key advantages of this automated approach are the ability to increase sample size, reduce variability during reprogramming or differentiation, and enable medium to high-throughput analysis of human PSCs and derivatives. These techniques will become increasingly important with the emergence of clinical trials using stem cells.

scholarly journals C-STEM: ENGINEERING NICHE-LIKE MICRO-COMPARTMENTS FOR OPTIMAL AND SCALE-INDEPENDENT EXPANSION OF HUMAN PLURIPOTENT STEM CELLS IN BIOREACTORS

2021 ◽  
Author(s):  
Philippe J.R. Cohen ◽  
Elisa Luquet ◽  
Justine Pletenka ◽  
Andrea Leonard ◽  
Elise Warter ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Large Scale ◽  
Scale Up ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Human Pluripotent Stem Cells ◽  
Cell Therapies ◽  
Cellular Source

Human pluripotent stem cells (hPSCs) have emerged as the most promising cellular source for cell therapies. To overcome scale up limitations of classical 2D culture systems, suspension cultures have been developed to meet the need of large-scale culture in regenerative medicine. Despite constant improvements, current protocols relying on the generation of micro-carriers or cell aggregates only achieve moderate amplification performance. Here, guided by reports showing that hPSCs can self-organize in vitro into cysts reminiscent of the epiblast stage in embryo development, we developed a physio-mimetic approach for hPSC culture. We engineered stem cell niche microenvironments inside microfluidics-assisted core-shell microcapsules. We demonstrate that lumenized three-dimensional colonies maximize viability and expansion rates while maintaining pluripotency. By optimizing capsule size and culture conditions, we scale-up this method to industrial scale stirred tank bioreactors and achieve an unprecedented hPSC amplification rate of 282-fold in 6.5 days.

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