Changes in Purine Metabolism During Differentiation of Dopamine Neurons from Human Induced Pluripotent Stem Cells

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Purines are a class of ubiquitous molecules required for fundamental processes in all cells. Purines are derived from two major sources: de novo synthesis, and salvage of preformed purine bases. The current studies provide evidence that the relative contributions of these two pathways change substantially as human induced pluripotent stem cells (iPSCs) differentiate into neurons. Expression of all genes in the de novo synthesis pathway decreases as pluripotent cells differentiate into neurons, but expression of the salvage pathway gene HPRT1 increases. This selective rise in HPRT1 gene expression corresponds with increased activity of its associated enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt). Similar changes in the expression of genes for the de novo pathway genes and for HPRT1 were found in a public database of gene expression for human brain development. The consequences of eliminating HGprt-mediated recycling were also evaluated in human-derived iPSCs with null HPRT1 mutations and stock iPSC lines that have been gene-edited to contain a null HPRT1 mutation. The absence of HGprt had no apparent impact on early neuronal differentiation, through 60 days of in vitro differentiation. Biochemical studies of purine content showed that that the absence of HGprt had little impact on intracellular purines, although large amounts of its substrate (hypoxanthine) accumulated in the tissue culture medium. Neurons derived from iPSCs without HGprt appeared morphologically and neurochemically indistinguishable from neurons derived from iPSCs where HGprt was intact. Interrogation of the transcriptome using RNA sequencing (RNAseq) indicated that the absence of HGprt had no consistent impact on gene expression during differentiation. Overall, these results suggest HGprt does not have a large impact on early neuronal differentiation and may instead play a more important role in later neuronal differentiation or function. Study design. The first study involved parallel differentiation of 4 CON (blue) and 4 LND (red) human-derived iPSC lines (HD-iPSCs) and collection of samples at 4 developmental stages. The second study involved 3 stock line iPSCs (SL-iPSCs) in blue (KOLF2.0, NCRM1, PGP1) each with a corresponding HPRT1 c.508C>T gene-edited subline (red), also sampled at 4 developmental stages. This figure was created in BioRender, Sutcliffe, D. (2026), agreement YH29LNHTYB, https://BioRender.com/mqw9vbc .

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