(108b) A Defined, Scalable 3D Culture System for Producing Human Pluripotent Stem Cells and Their Progeny

Introduction: Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), have the capacities for indefinite in vitro expansion and differentiation into presumably all cell types in the human body.  They therefore represent highly promising cell sources for numerous biomedical applications, such as cell replacement therapy, tissue and organ engineering, and pharmacology and toxicology screens.  Many such applications require large numbers of cells of high quality and purity.  For instance, ~10⁵ surviving dopaminergic neurons, ~10⁹ cardiomyocytes, or ~10⁹ β cells are required to treat a patient with Parkinson’s disease (PD), myocardial infarction (MI), or type I diabetes, respectively, and cell survival after engraftment is typically very low (~1%).  Analogously, ~10¹⁰ hepatocytes are needed for an artificial human liver, and ~10¹⁰ cells may be required to screen a million compound library.  Considering the large patient populations with degenerative diseases or organ failure (e.g. over 1 million with PD, 1-2.5 million with type I diabetes, ~8 million with MI, and 5.7 million with heart failure in the US alone), as well as the millions of chemical/peptide/nucleotide compounds that can be screened against thousands of molecular targets, massive numbers of hPSCs may be needed to deliver on the therapeutic promise of these stem cells.  It is becoming clear that the current 2D-based cell culture systems are incapable of producing sufficient cells with high quality and are becoming a bottleneck for these downstream applications.  In this presentation, we will introduce a simple, defined, scalable, GMP compliant 3D culture system for the production of hPSCs and their progenies. 

Results and Discussion: With this system, we have achieved long-term, serial expansion (> 60 passages) of multiple hPSC lines with a high replication rate (20-fold over 5-day per passage), yield (2.0 x10⁷ cells/ml), and purity (95% Oct4+), with single cell inoculation, all of which offer considerable improvements over the current approaches.  After long term culture in 3D, hPSCs remained pluripotent as shown by their ability to form all the 3 germ layers (ectoderm, mesoderm and endoderm) during the in vitro EB assay and in vivo teratoma assay.  More importantly, directed differentiation of hPSCs into their progenies (e.g. dopaminergic neuron progenitors) can be conducted efficiently following expansion, enabling us to produce large numbers of cells with therapeutic relevance. 

Conclusions:  In summary, we developed an efficient GMP compliant 3D system for producing hPSCs and their progeny.  This system will be of broad interest from the laboratory to the biotechnology scale.  In smaller scale, it can benefit animal studies or initial cell based screens.  In larger scale, it is GMP compatible and can aid biomedical efforts to develop cell therapy, artificial tissues, and high throughput drug screening with hPSCs.