Lineage restriction of human hepatic stem cells to mature fates is made efficient by tissue-specific biomatrix scaffolds.
Current protocols for differentiation of stem cells make use of multiple treatments of soluble signals and/or matrix factors and result typically in partial differentiation to mature cells with under- or overexpression of adult tissue-specific genes. We developed a strategy for rapid and efficient differentiation of stem cells using substrata of biomatrix scaffolds, tissue-specific extracts enriched in extracellular matrix, and associated growth factors and cytokines, in combination with a serum-free, hormonally defined medium (HDM) tailored for the adult cell type of interest. Biomatrix scaffolds were prepared by a novel, four-step perfusion decellularization protocol using conditions designed to keep all collagen types insoluble. The scaffolds maintained native histology, patent vasculatures, and ≈1% of the tissue's proteins but >95% of its collagens, most of the tissue's collagen-associated matrix components, and physiological levels of matrix-bound growth factors and cytokines. Collagens increased from almost undetectable levels to >15% of the scaffold's proteins with the remainder including laminins, fibronectins, elastin, nidogen/entactin, proteoglycans, and matrix-bound cytokines and growth factors in patterns that correlate with histology. Human hepatic stem cells (hHpSCs), seeded onto liver biomatrix scaffolds and in an HDM tailored for adult liver cells, lost stem cell markers and differentiated to mature, functional parenchymal cells in ≈1 week, remaining viable and with stable mature cell phenotypes for more than 8 weeks.


CONCLUSIONS
Biomatrix scaffolds can be used for biological and pharmaceutical studies of lineage-restricted stem cells, for maintenance of mature cells, and, in the future, for implantable, vascularized engineered tissues or organs.
Department of Cell and Molecular Physiology and Program in Molecular Biology and Biotechnology, UNC School of Medicine, Chapel Hill, NC 27599, USA.
Yunfang
Y
Wang
Cai-Bin
CB
Cui
Mitsuo
M
Yamauchi
Patricia
P
Miguez
Marsha
M
Roach
Richard
R
Malavarca
M Joseph
MJ
Costello
Vincenzo
V
Cardinale
Eliane
E
Wauthier
Claire
C
Barbier
David A
DA
Gerber
Domenico
D
Alvaro
Lola M
LM
Reid
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D019070:Cell Lineage; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism*; D016895:Culture Media, Serum-Free; D005109:Extracellular Matrix / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D008099:Liver / Q000166:cytology* / Q000378:metabolism; D010477:Perfusion / Q000379:methods; D051381:Rats; D013234:Stem Cells / Q000502:physiology*; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds
D016895:Culture Media, Serum-Free; D036341:Intercellular Signaling Peptides and Proteins; D003094:Collagen