We evaluated the potential of Fourier-transform infrared (FTIR) microspectroscopy for non-invasive biochemical profiling of rhesus macaque embryonic stem cells (rhESCs) cultured in either conventional FGF2/KOSR medium or a novel formulation, ALGöX. Cells from both conditions were analyzed by immunocytochemistry, RNA sequencing, and high-resolution FTIR profiling. Molecular marker expression patterns and transcriptional profiles revealed that rhESCs maintained in FGF2/KOSR were in the primed pluripotent state, whereas those cultured in ALGöX adopted a naïve-like state. FTIR spectra showed consistent differences in protein, lipid, and nucleic acid signatures, with ALGöX-cultured cells displaying higher amide I/II and nucleic acid absorbance and FGF2/KOSR-cultured cells exhibiting stronger lipid-associated bands. Principal component analysis (PCA) separated the two groups along PC−1 (64% variance), and partial least squares discriminant analysis (PLS-DA) classified samples with 100% specificity and 100% sensitivity. These findings demonstrate that FTIR microspectroscopy can reliably discriminate pluripotent state–specific biochemical features in non-human primate PSCs, providing a rapid and label-free approach for monitoring stem cell identity and quality.

We evaluated the potential of Fourier-transform infrared (FTIR) microspectroscopy for non-invasive biochemical profiling of rhesus macaque embryonic stem cells (rhESCs) cultured in either conventional FGF2/KOSR medium or a novel formulation, ALGöX. Cells from both conditions were analyzed by immunocytochemistry, RNA sequencing, and high-resolution FTIR profiling. Molecular marker expression patterns and transcriptional profiles revealed that rhESCs maintained in FGF2/KOSR were in the primed pluripotent state, whereas those cultured in ALGöX adopted a naïve-like state. FTIR spectra showed consistent differences in protein, lipid, and nucleic acid signatures, with ALGöX-cultured cells displaying higher amide I/II and nucleic acid absorbance, and FGF2/KOSR-cultured cells exhibiting stronger lipid-associated bands. Principal component analysis (PCA) separated the two groups along PC1 (64% variance), and partial least squares-discriminant analysis (PLS-DA) classified samples with 100% specificity and 100% sensitivity. These findings demonstrate that FTIR microspectroscopy can reliably discriminate pluripotent state–specific biochemical features in non-human primate PSCs, providing a rapid and label-free approach for monitoring stem cell identity and quality.