Process Analytical Utility of Raman Microspectroscopy in the Directed Differentiation of Human Pancreatic Insulin-Positive Cells

Abstract

Continued advances toward cell-based therapies for human disease generate a growing need for unbiased and label-free monitoring of cellular characteristics. We used Raman microspectroscopy to characterize four important stages in the 26-day directed differentiation of human embryonic stem cells (hESCs) to insulin-positive cells. The extent to which the cells retained spectroscopic features of pluripotent cells or developed spectroscopic features suggestive of pancreatic endocrine cells, as well as assessing the homogeneity of the cell populations at these developmental stages, were of particular interest. Such information could have implications for the utility of Raman microspectroscopy process analysis for the generation of insulin-positive cells from hESCs. Because hESC seeding density influences the subsequent pancreatic development, three different seeding density cultures were analyzed. Transcription factor and other marker analyses assessed the progress of the cells through the relevant developmental stages. Increases in the Raman protein-to-nucleic acid band ratios were observed at the final endocrine stage analyzed, but this increase was less than expected. Also, high glycogen band intensities, somewhat unexpected in pancreatic endocrine cells, suggested the presence of a substantial number of glycogen containing cells. We discuss the potential process analytical technology application of these findings and their importance for cell manufacturing.

A single-cell Raman-based platform to identify developmental stages of human pluripotent stem cell-derived neurons

“…As the change in glycogen content was significant in both SCRS and conventional assays, we suggest that glycogen can be used as a biomarker to distinguish hiPSCs from their differentiated neural progenies. Previously, evidence of glycogen variations has been reported during maintenance of human embryonic stem cells (hESCs) and hESC differentiation using Raman microspectroscopy ( 60 – 63 ). While glycogen increased during hESC differentiation into pancreatic insulin-positive cells ( 60 ), interestingly, our results demonstrated a different trend where a decrease of glycogen was found in differentiated neural lineages.…”

“…Previously, evidence of glycogen variations has been reported during maintenance of human embryonic stem cells (hESCs) and hESC differentiation using Raman microspectroscopy ( 60 – 63 ). While glycogen increased during hESC differentiation into pancreatic insulin-positive cells ( 60 ), interestingly, our results demonstrated a different trend where a decrease of glycogen was found in differentiated neural lineages. These inconsistent results on the glycogen concentration were more likely attributed to different cell lineages instead of differences between hESCs and hiPSCs ( 47 , 64 ), suggesting the importance of tracking the progression of lineage-specific differentiation and acknowledging the differences among cell types.…”

Identifying States along the Hematopoietic Stem Cell Differentiation Hierarchy with Single Cell Specificity via Raman Spectroscopy

“…17–19 The differentiation states of human embryonic stem cells and MSCs have also been identified based on combinations of Raman spectral features, such as differences in DNA-to-protein-related peaks. 15,18,20–27 Multivariate analysis has enabled monitoring stem cell differentiation and various differentiation-associated biomarkers based on changes over the cell fingerprint region in the Raman spectra. 21,23,25–29 Unlike MSCs, whose differentiation can be tracked by changes in endogenously produced biomolecules with readily identifiable Raman signatures, 17–19 such differentiation markers are not expected for HSCs.…”

Physico-electrochemical Characterization of Pluripotent Stem Cells during Self-Renewal or Differentiation by a Multi-modal Monitoring System

“…In this regard, a new quantitative analytical methodology enabling real-time observation of cellular behaviors in a non-destructive manner would advance our fundamental understanding in stem cell biology. Recently, Raman spectroscopy demonstrated its ability to assess the degree of stem cell differentiation by non-destructively detecting macromolecular compositional changes (Chan et al., 2009, Konorov et al., 2015, Schulze et al., 2010). However, such chemical changes are associated with protein expression, which typically occur at the later stages of stem cell differentiation.…”