title: 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.
authors.affiliation: Michael Smith Laboratories, The University of British Columbia , 2185 East Mall, Vancouver, BC Canada , V6T 1Z4.|Department of Chemistry, The University of British Columbia , 2036 Main Mall, Vancouver, BC Canada , V6T 1Z1.
authors.forename: Stanislav O
authors.initials: SO
authors.lastname: Konorov
authors.affiliation: Michael Smith Laboratories, The University of British Columbia , 2185 East Mall, Vancouver, BC Canada , V6T 1Z4.
authors.forename: H Georg
authors.initials: HG
authors.lastname: Schulze
authors.affiliation: Department of Cellular and Physiological Sciences, The University of British Columbia , 2350 Health Sciences Mall, Vancouver, BC Canada , V6T 1Z3.
authors.forename: Blair K
authors.initials: BK
authors.lastname: Gage
authors.affiliation: Department of Cellular and Physiological Sciences, The University of British Columbia , 2350 Health Sciences Mall, Vancouver, BC Canada , V6T 1Z3.|Department of Surgery, The University of British Columbia , 910 West 10th Avenue, Vancouver, BC Canada , V5Z 4E3.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Kieffer
authors.affiliation: Michael Smith Laboratories, The University of British Columbia , 2185 East Mall, Vancouver, BC Canada , V6T 1Z4.|Department of Chemical and Biological Engineering, The University of British Columbia , 2360 East Mall, Vancouver, BC Canada , V6T 1Z3.
authors.forename: James M
authors.initials: JM
authors.lastname: Piret
authors.affiliation: Department of Chemistry, The University of British Columbia , 2036 Main Mall, Vancouver, BC Canada , V6T 1Z1.
authors.forename: Michael W
authors.initials: MW
authors.lastname: Blades
authors.affiliation: Michael Smith Laboratories, The University of British Columbia , 2185 East Mall, Vancouver, BC Canada , V6T 1Z4.|Department of Chemistry, The University of British Columbia , 2036 Main Mall, Vancouver, BC Canada , V6T 1Z1.|Department of Electrical and Computer Engineering, The University of British Columbia , 2332 Main Mall, Vancouver, BC Canada , V6T 1Z4.
authors.forename: Robin F B
authors.initials: RF
authors.lastname: Turner
mesh_terms: D018929:Cell Culture Techniques / Q000295:instrumentation*; D002454:Cell Differentiation; D053595:Embryonic Stem Cells / Q000166:cytology; D006003:Glycogen / Q000032:analysis; D006801:Humans; D007328:Insulin / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000378:metabolism; D010179:Pancreas / Q000166:cytology*; D013059:Spectrum Analysis, Raman
chemical_list: D007328:Insulin; D006003:Glycogen