Nuclear Magnetic Resonance Metabolomic Footprinting of Human Hepatic Stem Cells and Hepatoblasts Cultured in Hyaluronan-Matrix Hydrogels

¹ Departments of Cell and Molecular Physiology and Biomedical Engineering, Program in Molecular Biology and Biotechnology, Cancer Center and Center for Gastrointestinal and Biliary Disease Biology (CGIBD), UNC School of Medicine, Chapel Hill, NC 27599
² Biomedical Engineering, UNC School of Medicine, Chapel Hill, NC 27599
³ Cancer Center and Center for Gastrointestinal and Biliary Disease Biology (CGIBD), UNC School of Medicine, Chapel Hill, NC 27599
⁴ Department of Medicinal Chemistry, Center for Therapeutic Biomaterials, 419 Wakara Way, Suite 205. Salt Lake City, Utah 84108

^* To whom correspondence should be addressed. E-mail: wsturner{at}email.unc.edu.

Correspondence may also be addressed to Lola Reid at lmreid@email.unc.edu

	Abstract

Human hepatoblasts, hHBs, and hepatic stem cells, hHpSCs, were maintained in serum-free Kubota's Medium, a defined medium tailored for hepatic progenitors, and on culture plastic versus within hyaluronan hydrogels mixed with specific combinations of extracellular matrix components (e.g. type I collagen and laminin). Nuclear magnetic resonance (NMR) spectroscopy was used to define metabolomic profiles for each substratum tested. The hHpSCs on culture plastic survived throughout the culture study, whereas hHBs on plastic died within 7-10 days. Both survived and expanded in all hydrogel-matrix combinations tested for more than 4 weeks. Profiles of hundreds of metabolites were narrowed to a detailed analysis of 8, such as glucose, lactate, and glutamine, shown to be significant components of cellular pathways including the Krebs and urea cycles. The metabolomic profiles indicated that hHpSCs on plastic remained as stem cells expressing low levels of albumin but no alpha-fetoprotein (AFP); those in hydrogels were primarily hHBs, expressing AFP, albumin and urea. Both hHpSCs and hHBs used energy provided by anaerobic metabolism. Variations in hyaluronan-matrix chemistry resulted in distinct profiles correlating with growth or with differentiative responses. Metabolomic footprinting offers non-invasive and non-destructive assessment of physiological states of stem/progenitor cells ex vivo.