Non-invasive monitoring of midbrain dopaminergic progenitor cell production from human pluripotent stem cells

Abstract

Abstract Background An emerging regenerative medicine for Parkinson's is cell replacement therapy with midbrain dopaminergic (mDA) progenitor cells produced from human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs). Although protocols are well described, the process of hESC/iPSC differentiation remains challenging. Furthermore, different clonal iPSC lines can have variable responses to the same mDA protocol conducted in the same laboratory. Methods One solution to address this issue is to identify secreted biomarkers that are predictive of mDA differentiation efficiency. We used candidate approaches and unbiased proteomic methods to interrogate the secretome of cells as they are converting from pluripotent cells to committed mDA progenitor cells. We used conditioned media from two laboratories with different mDA protocols (Edinburgh, Kyoto) and multiple pluripotent stem cell lines (RC17, MasterShef7, 1231A3, 404C2). Results We have identified a collection of secreted molecules, including TFF3, CORIN, PDGFC, SERPINF1, and NRP1, that increase in conditioned media during the early stages of mDA differentiation. The kinetics of up-regulation and abundance of secreted biomarkers exhibited unique signatures for each cell line. However, in all cases we could identify biomarker signatures as early as day 4 of mDA protocols that were predictive of mDA differentiation efficiency. We further examined the secretome of differentiating striatal and cortical neurons to discover biomarkers of non-mDA cells, which led to the identification of PLAU and NCAM1 as diagnostic markers that should remain low during mDA differentiation. Finally, we multiplexed selected positive and negative markers to construct custom Luminex assay systems that can non-invasively analyse media samples during the time-course of mDA progenitor cell production for five hESC/iPSC lines. Conclusion We have identified a collection of over 10 secreted biomarkers useful for non-invasively monitoring the production of mDA progenitor cells for day 4 of differentiation from multiple clonal pluripotent stem cell lines.