Open AccessAn allied reprogramming, selection, expansion and differentiation platform for creating hiPSC on microcarriers
Author(s) -
Lam Alan Tin Lun,
Ho Valerie,
Vassilev Svetlan,
Reuveny Shaul,
Oh Steve Kah Weng
Publication year - 2022
Publication title -
cell proliferation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.647
H-Index - 74
eISSN - 1365-2184
pISSN - 0960-7722
DOI - 10.1111/cpr.13256
Subject(s) - microcarrier , reprogramming , trypsinization , embryoid body , microbiology and biotechnology , induced pluripotent stem cell , biology , stem cell , cellular differentiation , haematopoiesis , chemistry , cell culture , embryonic stem cell , cell , biochemistry , genetics , gene , trypsin , enzyme
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.