Abstract

Human pluripotent stem cells (hPSC) are self-renewing cells having the potential of differentiation into the three lineages of somatic cells and thus can be medically used in diverse cellular therapies. One of the requirements for achieving these clinical applications is development of completely defined xeno-free systems for large-scale cell expansion and differentiation. Previously, we demonstrated that microcarriers (MCs) coated with mouse laminin-111 (LN111) and positively charged poly-l-lysine (PLL) critically enable the formation and evolution of cells/MC aggregates with high cell yields obtained under agitated conditions. In this article, we further improved the MC system into a defined xeno-free MC one in which the MCs are coated with recombinant human laminin-521 (LN521) alone without additional positive charge. The high binding affinity of the LN521 to cell integrins enables efficient initial HES-3 cell attachment (87%) and spreading (85%), which leads to generation of cells/MC aggregates (400 μm in size) and high cell yields (2.4–3.5×106 cells/mL) within 7 days in agitated plate and scalable spinner cultures. The universality of the system was demonstrated by propagation of an induced pluripotent cells line in this defined MC system. Long-term pluripotent (>90% expression Tra-1-60) cell expansion and maintenance of normal karyotype was demonstrated after 10 cell passages. Moreover, tri-lineage differentiation as well as directed differentiation into cardiomyocytes was achieved. The new LN521-based MC system offers a defined, xeno-free, GMP-compatible, and scalable bioprocessing platform for the production of hPSC with the quantity and quality compliant for clinical applications. Use of LN521 on MCs enabled a 34% savings in matrix and media costs over monolayer cultures to produce 108 cells.

Highlights

  • Human pluripotent stem cells such as human embryonic stem cells and human-induced pluripotent stem cells are expected to be used for cell-based therapeutics due to their ability for selfrenewal and the potential to develop into various cell types.[1]

  • Previously, we have shown that Human pluripotent stem cells (hPSC) can grow in continuously agitated conditions on PS MCs coated with positive charge and mouse LN111.5 In this work, we have explored the possibility of using higher affinity recombinant human LN521 (Kd = 0.72 – 0.22 nM, to integrin receptors9,19) as a sole coating of the MCs to develop a xeno-free, simpler, and scalable agitated culturing system

  • Progress has been made in developing defined xeno-free substrates for long-term hPSC self-renewal in defined media in conventional 2D monolayer plate (MNL) cultures.[2,18,21,34,35,36]

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Summary

Introduction

Human pluripotent stem cells (hPSC) such as human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC) are expected to be used for cell-based therapeutics due to their ability for selfrenewal and the potential to develop into various cell types.[1]. High volumetric cell yields are achieved due to better oxygenation, better metabolite mass transport, as well as limited microenvironment toxicity.[5,8] Recently, we described a defined, serum-free MC culture platform in which polystyrene (PS) MC are coated with cationic poly-l-lysine (PLL)

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