Introduction
In an attempt to better understand the ‘omics’ of stem cells, CHI’s 2nd annual Back to the Science of Stem Cell Research meeting focused on recent advances in the development of in vitro tissue models, and the potential applications of these systems to biomedical applications such as drug screening, diagnostics and therapeutics. Session topics at the meeting included: ‘Stem Cell Culture, Expansion, Sources’; ‘Exploring Alternative Therapeutics’; ‘Stem Cells: Culture and Creating 3D Models’; ‘Sources, Origins and Fate’; and ‘Exploring Screening Tools’. This report covers presentations from the final three session topics.
Mechanical stimuli in tissue formation
Cellular interactions are maintained more effectively in 3-D culture than in typical 2-D culture, and the rigidity of such 3-D systems is a critical feature regulating normal and abnormal tissue formation. In the opening plenary keynote presentation for the session on ‘Stem Cells: Culture and Creating 3D Models’, Valerie M Weaver (University of Pennsylvania, USA) described how mammary epithelial cells cultured in Matrigel became organized into acinar structures with hollow lumens. Matrigel has mechanical properties that are similar to mammary tissue, and altering its rigidity causes a loss of polarity, filling of the lumen and disruption of cell-cell junctions. Using synthetic materials, Dr Weaver demonstrated that rigid materials are able to produce mature focal adhesions that are not typically observed within softer materials, and that can increase the activity and duration of pathways such as extracellular signal-regulated kinase (ERK) and phosphatidylinositol (PI)3 kinase. The role of the mechanics of 3-D systems in the malignant transformation of cells is unclear; however, in vitro experiments suggest that, at a minimum, the matrix mechanics can cooperate with oncogene activation to promote migration from the structure to simulate malignant invasions. The continued use of these models may allow for an investigation into the mechanisms that are involved in cell transformation, and may ultimately identify targets for therapeutic intervention that are alternative targets to oncogenes.
Collagen and silk scaffolds
The fundamental design principles of scaffolds for cell growth remain poorly defined, but key components of these scaffolds include mechanics, chemistry and morphology. David Kaplan (Tufts University, USA) described the use of the fibrous proteins collagen and silk in several applications. The expansion of mesenchymal stem cells requires an increase in cell numbers without a loss in cellular potential. Stem cells that are cultured on denatured collagen maintain a potential for osteogenic differentiation that is similar to the potential for unexpanded stem cells. Silk proteins, which can form fibrous materials in a manner similar to collagen, have been used as aligned fibers for an artificial ligament, and as porous scaffolds for bone formation. Silk exhibits more stability than collagen in vivo, and therefore provides greater mechanical integrity over time. The silk proteins can also be genetically engineered with various functional groups. Fusion domains are incorporated into the sequence of the silk protein; these domains tend to be more hydrophilic than the native silk, and often are present at the surface of the material.
Technical versus commercial success in tissue engineering
In the past two decades, the resolution of several technical challenges has allowed for advances in guiding tissue formation. The technical successes in the field, however, do not always translate into commercial successes. Several tissue-engineering products are currently available, yet the development of additional products has remained a challenge. James Burns (Genzyme Biosurgery, USA) described Genzyme’s experience with Carticel for use as a cartilage replacement therapy. The Carticel approach involves a biopsy followed by cell isolation, de-differentiation and expansion for delivery into the cartilage defect, which is then covered by a periosteal flap. The treatment of several thousands of patients has indicated that this approach is successful, with a single application resulting in a patient satisfaction of almost 90%. However, Carticel is currently only used in approximately 1% of cartilage replacement cases. The challenges for this system are wide-ranging, and include aspects such as the relative complexity of the approach, significant capital investment, reimbursement from insurance companies and the regulatory environment for new therapies.
Challenges with telomere shortening in stem cells generation
Telomeres are repetitive DNA sequences and proteins that cap and protect chromosomes; the shortening of these telomeres leads to apoptosis. Stem cells that are generated by somatic cell nuclear transfer must recreate the telomere length of normal stem cells, or the appearance of premature aging may otherwise occur. Studies are being performed to characterize the mechanisms that are associated with telomere shortening. David Keefe (University of South Florida, USA) described how stem cells avoid senescence or death by lengthening telomeres using telomerase or telomere recombination. Techniques that are used to generate stem cells, such as somatic cell nuclear transfer and parthenogenic activation, can lead to telomere shortening. Telomerase activity is normally low in oocytes and embryos, but telomeres are observed to shorten abruptly during cleavage.
Primate ES cell research to optimize stem cell generation
Although embryonic stem (ES) cells are generally regarded as a future approach for many cell-based therapies, many questions remain regarding the production of these cells and the generation of patient-specific stem cells. Carol Brenner (University of New Orleans, USA) addressed the fundamental question of what is considered a ‘high quality’ embryo for stem cell generation. Research is being performed with nonhuman primates in which in vitro fertilization (IVF) is routinely performed and for which in vivo-produced embryos can be obtained for comparison. Procedures such as follicle stimulating hormone/human chorionic gonadotropin stimulation can affect deletions of the mitochondrial DNA within the oocyte, and may affect the integrity of chromosomal DNA.
ES cells in high-throughput screens
Cell-based assays are increasingly being used for drug screening, and the source of cells is recognized as a critical component of any such screen. Marsha Roach (Pfizer Global Research & Development, USA) discussed the advantages of using ES cells as the cell source in screening applications. Relative to cell lines or primary cell isolations, stem cells have a normal genetic structure, and have growth patterns that provide an almost limitless supply of cells. In addition, these cells can be developed for virtually any tissue, and have more consistent responses, which are expected to be invariant with long-term culture. ES cells can be differentiated to a neuronal lineage and be used to screen ligands for activity through a specific receptor, which is expressed endogenously, rather than by forced expression through an exogenous gene. These cells can be differentiated efficiently to a neuronal lineage, and have functional synaptic activity through multiple receptor systems. The differentiated cells have been demonstrated to tolerate the solvents that are used for drug solubilization, and can respond to the agonists and antagonists that are typically used with primary neurons.
Squamous cell carcinoma progression
Jonathan Garlick (Tufts University, USA) described the use of 3-D models of cell growth as systems to test hypotheses regarding basic mechanisms of precancer to cancer development, to perform drug screening, and to validate targets for therapy. The culture system involved the co-culturing of cells on collagen at an air/water interface, with an intact basement membrane at one face of the collagen scaffold. Cancer cells were mixed with normal cells to provide foci from which invasive cancer could progress. The progression was characterized into four model environments: intraepithelial dormancy, transepithelial migration, basement membrane adhesion and incipient invasion. The presence of normal cells can suppress the malignant phenotype, although some cellular insults can overcome this suppression. The loss of Epithelial-cadherin, for example, increases signaling activity through several integrins and leads to greater migration of cancer cells through the tissue. The 3-D model systems of cell growth may ultimately be useful in developing personalized therapies by employing cells from primary tumors.
Ovarian follicle culturing
Traditional IVF has been a successful approach for infertility research, yet it is not appropriate for research in certain cases, such as for hormone-responsive cancers and childhood cancers. Lonnie D Shea and Teresa K Woodruff (both Northwestern University, USA) developed a 3-D culture system for immature mouse ovarian follicles that are capable of producing mature oocytes. The oocytes that are retrieved following in vitro culture have properties that are similar those of in vivo-matured oocytes, producing comparable fertilization and live birth rates. The culture system is dependent on the initial stage of the follicle, and therefore has an impact on the required media supplements and the mechanical properties of the matrix. In addition to fertility preservation, this 3-D culture system could be employed with drug screening to identify compounds that promote follicle maturation, or to screen environmental toxins that diminish fertility.
Immunogenicity: A human lymph node model
Cell, drug and material interventions, virtually all of which are immunomodulatory, may influence the efficacy of a given stem cell treatment. Christoph Giese (ProBioGen AG, Germany) described the development of a human lymph node model for potential use in the generation of antibodies and antigen-specific cells and for drug screening. The model comprises a co-culture of dendritic cells and T- or B-cells that form microorganoids, and can provide readouts on cytokine release patterns, cell differentiation and cell organization. Cultures are generated in a bioreactor, and the media are sampled to determine cytokine release profiles. The use of this system was demonstrated by the process of determining the quantities of six different cytokines during 2 weeks of culture, with the production profile being dependent on the antigen being presented.
High-throughput approaches for biomaterials
High-throughput biomaterial synthesis and testing provides a complementary approach for identifying biomaterials that are capable of promoting desired responses. Daniel G Anderson (MIT, USA) described two applications for HTS: biomaterials for cell growth and gene delivery. Synthesis is performed in nanoliter volumes using acrylate groups on various compounds to create a robust system that is capable of diverse polymer formation. To identify biomaterials for cell growth, cells are cultured on the deposited materials and are stained for specific markers by immunohistochemistry, which can be analyzed with an automated process. This approach seeks to identify the appropriate combination of materials and growth factors that are required to produce a desired response. A similar approach has been used for the synthesis of gene therapy vectors. Several polymers have been identified that can produce similar levels of expression to the most common cationic polymer, polyethyleneimine, but with reduced cytotoxicity. Second-, third- and fourth-generation polymers have been identified and are currently being tested both in vitro and in vivo; preliminary studies indicate that polymers have been identified with high activity following direct injection in vivo.
Summary
The Back to the Science of Stem Cell Research meeting covered a broad array of approaches for the creation of cell and tissue models. These models can be employed in a variety of applications ranging from in vitro screening to in vivo tissue repair. Biomaterials, identified through either rational design or HTS, provide a central technology for the generation of 3-D tissue that is composed of multiple cell types. The presentations at the meeting highlighted the successes and current questions in the field of tissue engineering, and emphasized that many challenges remain in translating technical successes into commercial successes. Commercial benefit will depend not only on technical achievements, but also on more practical considerations that are associated with identifying the best opportunities for stem cell research and navigating the regulatory and insurance environment related to stem cell technologies.