Randall McClelland

Human hepatic stem cells from fetal and postnatal donors

Human hepatic stem cells (hHpSCs), which are pluripotent precursors of hepatoblasts and thence of hepatocytic and biliary epithelia, are located in ductal plates in fetal livers and in Canals of Hering in adult livers. They can be isolated by immunoselection for epithelial cell adhesion molecule–positive (EpCAM+) cells, and they constitute ∼0.5–2.5% of liver parenchyma of all donor ages. The self-renewal capacity of hHpSCs is indicated by phenotypic stability after expansion for >150 population doublings in a serum-free, defined medium and with a doubling time of ∼36 h. Survival and proliferation of hHpSCs require paracrine signaling by hepatic stellate cells and/or angioblasts that coisolate with them. The hHpSCs are ∼9 μm in diameter, express cytokeratins 8, 18, and 19, CD133/1, telomerase, CD44H, claudin 3, and albumin (weakly). They are negative for α-fetoprotein (AFP), intercellular adhesion molecule (ICAM) 1, and for markers of adult liver cells (cytochrome P450s), hemopoietic cells (CD45), and mesenchymal cells (vascular endothelial growth factor receptor and desmin). If transferred to STO feeders, hHpSCs give rise to hepatoblasts, which are recognizable by cordlike colony morphology and up-regulation of AFP, P4503A7, and ICAM1. Transplantation of freshly isolated EpCAM+ cells or of hHpSCs expanded in culture into NOD/SCID mice results in mature liver tissue expressing human-specific proteins. The hHpSCs are candidates for liver cell therapies.

Gradients in the liver's extracellular matrix chemistry from periportal to pericentral zones: influence on human hepatic progenitors.

Embryonic mesenchymal feeders produce paracrine signals requisite for ex vivo survival and expansion of hepatic progenitors. The signals consist of a subset of soluble factors found in conditioned medium, and a subset of insoluble factors found in extracellular matrix that include collagens and basal adhesion molecules. We have identified key matrix components required for ex vivo maintenance of human hepatic progenitors produced by biologically active feeders. These components are similar to those found in zone 1 of the liver acinus (e.g., space of Disse) between layers of parenchyma and endothelia. Within these layers are transition chemistry matrix gradients, from zone 1 to zone 3. Use of purified zone 1 matrix components enables attachment and expansion of human hepatic progenitors independent of feeders. Cells aggregated into spheroid-like structures on laminin or spread into monolayers on type III or IV collagens. Contrastingly, a zone 3 matrix component, type I collagen, elicited growth arrest and differentiation. Another zone 3 matrix component, fibronectin, inhibited attachment. Use of specific matrix components, along with soluble paracrine signals from feeders, should enable one to maintain hepatic progenitors ex vivo without feeders and under wholly defined conditions.

Hepatic Stem Cells and Hepatoblasts: Identification, Isolation, and Ex Vivo Maintenance

Publisher Summary This chapter discusses hepatic stem cells (HpSCs) and provides protocols on HpSCs, especially human hepatic stem cells (hHpSCs). It also includes development of a serum-free, hormonally defined medium (HDM), preparation of tissue extracts enriched in extracellular matrix, and methods to design biodegradable, polylactide scaffoldings or microcarriers in ways appropriate for progenitors and use of bioreactors. There has been recognition that the epithelial–mesenchymal relationship is lineage dependent. Epithelial stem cells are partnered with mesenchymal stem cells, and their differentiation is co-ordinate. In the liver, the lineages begin with the HpSCs paired with their mesenchymal partners and angioblasts that interact with multiple forms of paracrine signals. These two give rise to descendents in a stepwise, lineage-dependent fashion and their descendents remain in a partnership throughout differentiation. Tissue engineering involves the mimicking of the livers epithelial–mesenchymal relationship with recognition of the lineage-dependent phenomena. Serum-free, HDM have been found to select for parenchymal cells even when the cells are on tissue culture plastic. Tissue-specific gene expression is improved in cultures under serum-free conditions and especially with serum-free medium supplemented with only the specific hormones needed to drive expression of a given tissue-specific gene.

Successful transplantation of human hepatic stem cells with restricted localization to liver using hyaluronan grafts

Cell therapies are potential alternatives to organ transplantation for liver failure or dysfunction but are compromised by inefficient engraftment, cell dispersal to ectopic sites, and emboli formation. Grafting strategies have been devised for transplantation of human hepatic stem cells (hHpSCs) embedded into a mix of soluble signals and extracellular matrix biomaterials (hyaluronans, type III collagen, laminin) found in stem cell niches. The hHpSCs maintain a stable stem cell phenotype under the graft conditions. The grafts were transplanted into the livers of immunocompromised murine hosts with and without carbon tetrachloride treatment to assess the effects of quiescent versus injured liver conditions. Grafted cells remained localized to the livers, resulting in a larger bolus of engrafted cells in the host livers under quiescent conditions and with potential for more rapid expansion under injured liver conditions. By contrast, transplantation by direct injection or via a vascular route resulted in inefficient engraftment and cell dispersal to ectopic sites. Transplantation by grafting is proposed as a preferred strategy for cell therapies for solid organs such as the liver. (HEPATOLOGY 2013)

In Situ Labeling and Magnetic Resonance Imaging of Transplanted Human Hepatic Stem Cells

PurposeThe purpose is to address the problem in magnetic resonance imaging (MRI) of contrast agent dilution.ProceduresIn situ magnetic labeling of cells and MRI were used to assess distribution and growth of human hepatic stem cells (hHpSCs) transplanted into severe combined immunodeficiency (SCID)/non-obese diabetic (NOD) mice. It was done with commercially available magnetic microbeads coupled to an antibody to a surface antigen, epithelial cell adhesion molecule (EpCAM), uniquely expressed in the liver by hepatic progenitors.ResultsWe validated the microbead connection to cells and related MRI data to optical microscopy observations in order to develop a means to quantitatively estimate cell numbers in the aggregates detected. Cell counts of hHpSCs at different times post-transplantation revealed quantifiable evidence of cell engraftment and expansion.ConclusionsThis magnetic labeling methodology can be used with any antibody coupled to a magnetic particle to target any surface antigen that distinguishes transplanted cells from host cells, thus facilitating studies that define methods and strategies for clinical cell therapy programs.

7 – TISSUE ENGINEERING

Publisher Summary Tissue engineering is a biomedical engineering discipline that integrates biology with engineering to create tissues or cellular products outside the body or to make use of gained knowledge to better manage the repair of tissues within the body. Many new cellular therapies are being developed that create challenges for engineering tissue function. The implementation of cell therapies and grafts in the clinic requires the recognition and resolution of several difficult issues. These include tissue harvest, cell processing and isolation, safety testing, cell activation or differentiation, assay and medium development, storage and stability, and quality assurance and quality control issues. Furthermore, for device consideration—such as bioreactors—the function, choice, manufacturing, and treatment of biomaterials for cell growth and device construction are important. Systems analysis of metabolism, cell–cell communication, and other cellular processes also play key roles in replicating normalcy and defining bioartificial organ specifications. Finally, to successfully understand tissue function, it must be possible to quantitatively describe the underlying cellular fate processes and manipulate them.

Construction of a multicoaxial hollow fiber bioreactor.

Bioreactors are assembled tools conceived to exploit engineering principles with inbuilt biological -relevance. Such reactors are created as in vitro models to better replicate natural in vivo organs. These biotools are subsets within the interdisciplinary tissue engineering field and are established as inert devices to improve upon biological stimuli while simultaneously allowing tissue functional properties to be nondestructively measured. Design and fabrication efforts are focused on two-dimensional (2D) and three-dimensional (3D) physical constructs while linking environment-cell relations, the microenvironment. Product proficiencies generally involve material scaffolds, nutrient dispersion, compartmentalized units, passive and kinetic flow channels, temperature regulation, pressure management, and cell line or primary cells from assorted organs as tissues. Bioreactor advancements continue with interdisciplinary principles such as energy conservation, cell ecosystems, system-biological approaches, and viable-cell design innovation. Herein, we describe the design and construction of a hollow fiber multicoaxial bioreactor with integral oxygenation (i.e., oxygenation within the bioreactor proper) for use with liver cells, but it could be used with any anchorage-dependent cell type.