October 1, 2014 (Vol. 34, No. 17)

Just about everything to do with stem cells—from basic research to clinical development—will be discussed at the World Stem Cell Summit 2014, which will be held December 3–5, in San Antonio, TX.

This event is expected to highlight the use of stem cells in disease models for drug discovery; the innovative technologies, products, and services advancing regenerative medicine; the emerging role of regenerative medicine in clinical practice; the need (still unmet) for cell standardization; and the integrity of basic research and clinical development.

Evan Y. Snyder, M.D., Ph.D., professor at the Sanford-Burnham Medical Research Institute in La Jolla, CA, and director of its Center for Stem Cells and Regenerative Medicine and its Stem Cell Research Center and Core Facility, expects several emerging areas to be in the spotlight throughout the meeting. One is the increasing focus on using stem cells to model diseases for the purpose of drug discovery. The cell lines derived from stem cells are used to develop assays and screen compound libraries for drugs that alter disease-related cellular signatures.

Differentiation Protocols

A particularly challenging aspect of this work is differentiating the stem cells to obtain the specific cell type related to the disease in question so that effective drugs can be identified. This requires understanding the underlying pathology of the disease and being able to control the differentiation process. A drug screening assay is intended to predict as closely as possible what will happen in a patient, and the trend is toward screening in more specified cell types.

“Differentiation protocols are becoming more specialized and sophisticated,” says Dr. Snyder. For example, investigators that mean to target a disease that affects the nervous system may find that simply screening compounds against a generic neuron no longer suffices. It may, Dr. Snyder explains, be necessary to make “a motor neuron, an A9 as opposed to an A10 domaninergic neuron, or an upper layer cortical neuron versus a lower layer cortical neuron.”

Thomas P. Zwaka, M.D., Ph.D., a professor at the Black Family Stem Cell Institute at the Icahn School of Medicine at Mount Sinai, is experimenting with a new approach to generate more authentic disease models using induced pluripotent stem cells (iPSCs). The method is an alternative to the conventional approach described as “directed differentiation,” intended to guide stem cells along a directed path to form a particular tissue or structure. The new strategy is based on the premise that “cells are a lot smarter than we think,” remarks Dr. Zwaka.

Trying to orchestrate development in a petri dish is doable to a certain extent, he says, but requires a lot more knowledge than we currently have about developmental biology. Past efforts to generate cells from hematopoietic-derived iPSCs have not been able to produce cells that functionally mimic their intended targets when transplanted into mouse models or humans.

Yet pluripotent cells injected into an immunocompetent animal can form teratomas, which contain cell types from all over the body and are capable of forming highly organized structures such as hair follicles, even though these cells do not go through embryonic development. “It has not  been appreciated enough that cells are programmed to self-organize into organ structures,” Dr. Zwaka insists.

In his lab, researchers are trying to provide the correct in vitro environment to stimulate iPSCs to self-organize into neural tube-like structures. Instead of guiding the cells down a particular path, the approach involves providing an extracellular matrix comprised of fibronectin to encourage the cells to aggregate, adding various growth factors, and then disturbing the cells as little as possible.

The main challenge lies in biasing the process toward the formation of a particular lineage—in this case neurons and the formation of midbrain organoids—and determining when and how to bias the process while minimally manipulating the cells and closely mimicking normal development.

Dr. Zwaka’s goal is to make tyrosine hydroxylase-positive dopaminergic neurons that can be used for modeling Parkinson’s disease, for studying normal biology and developmental processes at the molecular level, and for developing stem cell therapies. This new approach, according to Dr. Zwaka, is needed “to prove that our product is more authentic” than iPSC-derived neuronal tissue generated using existing methods.

Technology, Tools, Techniques

An important topic of discussion in the area of stem cell transplantation is the benefits of developing “complex, multiple-cell-type graft materials to be able to tap into multimodal actions of a stem cell,” says Dr. Snyder. This builds on the recognition that in the setting of transplantation and tissue repair, stem cells engage in cross-talk and can provide functions other than cell replacement. Transplanting a range of cell types could take advantage of the cells’ ability to secrete beneficial substances including anti-inflammatory agents, for example.

The stem cell research community also continues to struggle with the many challenging questions that arise during clinical development. Such questions—When is a stem cell therapy ready for a clinical trial? How should a clinical trial be designed and structured? (For example, should it include a sham/control arm?) Who should receive a new therapy? (For example, should they be patients with early-stage disease who are more likely to benefit, or patients with late-stage disease for whom standard treatments have failed?)—can be answered only after researchers confront a host of technical, ethical, and regulatory issues.

Another area of intense focus involves biomaterial engineering and efforts to combine stem cells with various types of biomaterials to create three-dimensional complexes ex vivo for transplantation. For example, researchers are developing pre-engineered structures to grow stem cells outside the body for use as three-dimensional model systems that more accurately mimic real tissues in structure and function.

Dr. Snyder also anticipates that the Summit will include presentations by researchers focusing on the role of stem cells in cancer, including the latest findings suggesting that cancerous transformations initiate in stem cells. Such findings raise the possibility of targeting stem cells to attack cancer in its earliest stages. Also, stem cells could be used to deliver oncocidal factors to cancers by an activity called “oncotropism (a strategy already in clinical trials).

Commenting on the technology that has helped advance the stem cell field over the past 6–12 months, Graham C. Parker, Ph.D., a professor at The Carman and Ann Adams Department of Pediatrics, Wayne State University School of Medicine, and editor in chief of the journal Stem Cells and Development, highlights the application of novel genomic editing tools, allowing for “more rigorous and dependable manipulations of stem cell populations, whether it be to develop therapeutic screens or translational tools or to advance the study of development.”

As an example, he cites a Stem Cells and Development article that appeared online July 30, 2014. The article, by Kim et al., contends that “successful characterization of a cellular model of disease depends on your ability to minimize the genetic variance between the manipulated and the control cell samples. Up until quite recently, researchers have been happy to be able to create a population of cells that has the disease, but now they have the tools to create the appropriate control population for comparison.”

Jim Reid, chairman and chief executive officer of Sistemic, plans to present data on two new areas of research: 1) establishing a standard for mesenchymal stem cells (MSCs) and a guide that can be used to benchmark MSC products and 2) using microRNA (miRNA) profiles to predict cell outcomes, including potential clinical outcomes.

Sistemic analyzes the information contained in miRNA and other noncoding RNAs for use in the development of cell therapies and drug repositioning. One of the things missing from the current “stem cell toolbox” is “a better understanding of the characteristics needed for donor selection, be this in autologous or allogeneic therapies, and the development of biologically relevant potency and release assays,” says Reid.

He expects the progress made over the past 6–12 months in advancing products to the market with strong clinical data to be an important area of discussion at the upcoming Summit. “Additionally, big pharma seems to be becoming interested in the field and making investment commitments,” Reid adds. He also highlights the recent report on the development of a functioning thymus at the University of Edinburgh. “The link with academic research and the ability to translate this will be key,” he says.

Other emerging topics of debate and interest in the field include a comparison of pluripotent cells derived via somatic cell nuclear transfer (SCNT) and iPSCs and a determination of which resemble more closely embryonic stem cells, and which derivation process more efficiently erases the cells’ memory of their parental cell line. Speakers at the Summit are also likely to focus on efforts to identify alternate sources for pluripotent cells, beyond the preimplantation embryo, such as the development of amniotic stem cells.

Stem Cell Therapies

Mary Pat Moyer, Ph.D., founder, CEO, and CSO of San Antonio, TX-based Incell will not have to travel far to speak at this year’s World Stem Cell Summit, where she will talk about the complexities of managing the production of a stem cell product, from the isolation and expansion of cells through to an end-product, in a GMP setting with the necessary quality controls. Incell draws on years of experience building capabilities across multiple tissues, including adipose, skin, liver, and birth tissues, and developing the media, solutions, cryostorage, and other support components needed to build its contract services and internal manufacturing capabilities.

Dr. Moyer will also provide an update on the various clinical trials in which Incell is involved, including one set to begin later this year that will evaluate cell-enriched fat grafting for soft tissue repair in collaboration with the military medicine community. She attributes the growing interesting in using adipose tissue as a source of stem cells to the relative ease of isolating large numbers of cells and the available technology for isolating these cells.

Incell will manufacture the autologous cell therapy product for the planned trial. If the approach is successful, Dr. Moyer expects an allogeneic adipose-derived stem cell therapy for soft tissue repair to follow. She emphasizes the particular advantages of MSCs derived from adipose tissue, including their ability to mediate inflammatory processes and to promote microvascular repair, and their propensity to migrate to the sites where they are needed, enabling both local and systemic options for therapeutic products.

One area in which stem cell therapeutics production could clearly improve is greater recognition overall that “this is a manufacturing process that requires quality controls for product release,” says Dr. Moyer. Scale-up methods in particular need greater analytical standardization and quality control, in Dr. Moyer’s view.

Carrying cultured cells through multiple passages and scaling up to larger and larger cell numbers can alter cells, resulting in loss of biomarkers characteristic of stem cells and even loss of certain functionality. In essence, the stem cells a manufacturer starts out producing for research or preclinical use, or provides for a Phase I clinical trial, may differ from those ultimately administered to patients, or that are later produced in much larger quantities for Phase III clinical studies.

Adipose-derived mesenchymal stem cells in culture. The cells have DAPI-stained blue nuclei and express the CD44 cell surface biomarker (green fluorescence). Cells are growing in colonies and on the plastic substrate monolayer. [Incell Corp]

A Rapidly Evolving Field

What better place than a gathering of leading stem cell researchers to explore ways to disseminate research results related to regenerative medicine in a responsible and timely manner, helping to improve accountability and expedite access to key findings? Dr. Parker is looking forward to having a “full and frank discussion on what works and what perhaps could be improved in the current publication model.”

In Dr. Parker’s view, the ultimate aim is “to allow a greater transparency of the research efforts that will not only encourage greater public awareness of and investment in stem cell research, but also make it a more efficient enterprise that will reach the shared goals of academic understanding as well as therapeutic development in a more expeditious and veracious manner.”

An ongoing, open, and productive discussion about the directions and progress of stem cell research and its applications in regenerative medicine is essential, and too many scientists do not accept the ability (and value) of well-informed scientists and nonscientists alike scrutinizing their work and voicing their concerns, whether or not those concerns prove justified.

“The integrity of the enterprise and its potential to impede our progress are the biggest concerns,” Dr. Parker emphasizes.

Cell Lines Derived from Stem Cells Model Parkinson’s Disease

A little over two years ago, Life Technologies (now part of Thermo Fisher Scientific) and the Parkinson’s Institute initiated a collaborative program to develop induced pluripotent stem cell (iPSC)-based disease models. These models were derived from donor fibroblasts collected at the Institute.

In the early stages of the project, Thermo Fisher Scientific mainly used its TALEN™ technology to edit the genomes of the iPSCs derived from patients with Parkinson’s disease and correct two disease-relevant mutations: modifying the leucine-rich repeat kinase-2 (LRRK2) gene and the glucocerebrosidase gene to correct the Parkinson’s-related mutations in each and to revert the cell lines containing these genes to a wild-type genotype.

With the emergence of CRISPR technology for targeted gene editing, Thermo Fisher Scientific has paired mRNA-based CRISPR tools with its Lipofectamine® MessengerMAX™ Transfection Reagent to enhance the delivery of GeneArt® CRISPR Cas9 Nuclease mRNA into the iPSCs and improve the efficiency of the genome editing process.

Thermo Fisher Scientific has delivered the stem cell lines to the Parkinson’s Institute, which is using the model systems to study the molecular basis of the disease, with a particular interest in understanding environmental stressors that can trigger Parkinson’s in individuals who have a genetic predisposition. Thermo Fisher Scientific is also studying the cell lines internally, looking, for example, at the phenotypic differences between cells with the LRRK2 mutation and those in which the mutation has been corrected.

“Under certain conditions, cells with the LRRK2 mutation are more sensitive to stressors, and we can reverse that phenotype with the corrected gene or with a small molecule inhibitor,” says Kurt Vogel, Ph.D., associate director of external R&D at Thermo Fisher Scientific.

Another aspect of the partnership has focused on multiple systems atrophy (MSA), a sporadic form of Parkinson’s disease with no known genetic cause. Thermo Fisher Scientific has created knockout gene models using patient-derived cells, disabling one or both copies of the alpha-synuclein gene to inhibit the formation of alpha-synuclein protein-containing Lewy bodies, which accumulate in the brains of patients with MSA.

“The project has achieved its original goals,” remarks Dr. Vogel, with the production of the necessary tools and iPSC lines for modeling Parkinson’s and MSA. The collaboration is ongoing, with the evaluation of disease-relevant phenotypes using these cell lines. “The next stages would involve making dopaminergic neurons and looking at the phenotypes in those cells, and looking at other cell types relevant to MSA,” states Dr. Vogel.

Stem cells show promise in many research areas, especially in disease modeling. In 2013, Life Technologies announced a partnership with the Parkinson’s Institute in Sunnyvale, CA, to develop Parkinson’s disease model systems using fibroblasts from donor skin biopsies to generate induced pluripotent stem cells and edit the genome using company tools. The collaborators’ approach to modeling neurodegenerative disease is detailed in three white papers. Available online, these papers cover the generation of iPSCs, the generation of neural stem cells, and genome editing.

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