Cellular Reprogramming: Opportunities and Challenges

In 2007, researchers created the first human induced pluripotent stem cells (iPS cells) by infecting skin cells with four transcription factors (Oct-4, Klf4, Sox-2, and c-Myc).1 After the cells had achieved an induced pluripotent state, the researchers were able to differentiate them into new cell types. The ability to revert somatic cells to an embryonic state and then differentiate them into desired lineages offers a wealth of opportunities for disease research.

In addition to their potential as therapies based on cell replenishment, iPS cells are a novel tool for in vitro disease modeling and drug screening. As more and more researchers incorporate iPS cells into their studies, the technology to create and characterize them continues to be refined while new questions and challenges are revealed.

Evolving technology

Initial efforts to generate iPS cells required simultaneous coinfection of cells with four separate retroviral expression vectors. Each vector carried one transcription factor, which resulted in a high number of genomic integrations. Alternative approaches to iPS generation have included use of plasmids and nonintegrating adenovirus vectors to deliver the transcription factors. The rates at which cells convert to pluripotency using these methods, however, are far lower than those obtained using retroviral vectors.2

Figure 1 - Mouse iPS cell morphology and marker expression. Mouse embryonic fibroblasts (MEFs) infected with the STEMCCA lentivirus display characteristic ES cell morphology in a phase contrast image of a single mouse iPS cell colony seven days after infection (a). Passage 3 mouse iPS cells exhibited high alkaline phosphatase activity (b) and express high levels of Oct-4 (c), SOX-2 (d), and SSEA-1 (not shown). Cell nuclei were counterstained with 4ʹ,6-diamidino-2-phenylindole (DAPI) (blue).

Generation of human and mouse iPS cells can now be accomplished using a single, excisable polycistronic lentiviral vector that delivers all four Yamanaka transcription factors (STEMCCA™, EMD Millipore [Billerica, MA]; Figure 1). Use of a single vector significantly reduces the number of viral integrations required—in some cases, iPS clones possessing only a single viral integrant can be isolated.3

In addition to fully reprogrammed cells, transcription factor-induced reprogramming can generate undifferentiated cells that are not completely pluripotent.1,4 These partially reprogrammed cells (pre-iPS or Class I iPS cells) have global gene expression and DNA methylation patterns distinct from embryonic stem (ES) cells, despite similarities in colony morphology and the ability to propagate extensively in culture. Partially reprogrammed cells can be characterized by: continued expression of the viral transgenes; incomplete expression of pluripotent genes such as Nanog, SSEA-4, and TRA-1-60 with human cells; down-regulation of somatic cell marker genes; and in mouse cells, the inability to generate germ line transmitting chimeric mice.

A range of kits and reagents are available to assist researchers in their characterization of iPS cell cultures. Access to optimized tools and protocols can help accelerate research as more laboratories that are new to the stem cell field begin to work with iPS cells.

Figure 2 - a) Anticipated gene expression pattern of viral transgenes and Nanog, a marker of pluripotency for different cell types. b) Relative mRNA copy numbers for viral and Nanog genes normalized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression levels. Relative mRNA copy numbers of viral and Nanog genes from human iPS cells were plotted together with those from negative control (somatic cells) and positive control for viral gene (HEK293 cells transfected with OKSM viral vector) and human Nanog gene (Hu ESC).

Assessment of whether an iPS clone is a fully or partially reprogrammed colony can be rapidly accomplished using a multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR) kit (STEMCCA Viral Gene Detection, EMD Millipore). The kit detects the expression of the STEMCCA viral transgenes and ES/iPS cell pluripotent markers. Because the STEMCCA lentivirus is a single polycistronic vector containing four transcription factors, all the transgenes are transcribed as a single mRNA molecule. Subsequent transgene analysis shows the gene expression level of the entire cassette (Figure 2). In a fully reprogrammed colony, one would expect to see the down-regulation of viral transgene expression and the up-regulation of Nanog gene expression, a pluripotency marker.

A rapid, efficient flow cytometry-based process originally developed to characterize human ES cells can also be used to evaluate the expression of markers reflecting the degree of pluripotency of iPS cell lines. The FlowCellect hESC Characterization Kit (EMD Millipore) contains validated fluorescent antibodies to Oct-4; SSEA-4; and SSEA-1, a marker for monitoring transition of undifferentiated stem cells to a differentiated state. The labeled antibodies are optimized for cytometric analysis on the guava EasyCyte™ benchtop flow cytometer (EMD Millipore).

Typical cytometric results show iPS cell cultures having a distinct Oct-4 and SSEA-1 positive population of cells with significant separation from parent fibroblasts. Pluripotency is indicated by a shift in fluorescence intensity resulting from an increase in the expression of Oct-4 and SSEA-4, which are markers of acquired pluripotency. In contrast, the population of iPS cells expressing SSEA-1, a marker that coincides with human embryonic stem cell differentiation, is reduced. This same cytometric technique can also be used to compare different clones of the same iPS cell line.

Most recent efforts to reprogram human somatic cells to iPS cells utilize synthetic mRNAs encoding the four Yamanaka factors, which overcomes the problems associated with genomic integration and insertional mutagenesis.5

Disease modeling

iPS cells provide the ability to recapitulate both normal and pathological tissue formation in vitro, and can yield a genetically diverse set of patient- and disease-specific cells. Following well-established protocols, a disease researcher can take a skin biopsy from a patient and reprogram the isolated fibroblasts into iPS cells. Those iPS cells can then be differentiated into the cell types that are affected in the disease being studied, given the appropriate culture conditions.

A barrier to realizing the full potential of iPS cells is the ability to direct their differentiation into the cell types of interest. Identifying the right cocktail of media conditions, supplements, and growth factors that successfully drive iPS cells toward a desired lineage on a reproducible basis is a time-consuming, iterative exercise. A carefully choreographed series of signals must be recreated to guide cells down the chosen pathway. This labor-intensive work has already been done for a number of cell types. Kits and media containing an optimized set of factors necessary to differentiate stem cells to a chosen lineage are commercially available for generating neurons, oligodendrocytes, mesenchymal cells, and osteocytes.

iPS technology enables researchers to culture cell types that would normally be a challenge to access, such as those affected by neurological disorders. Diseases with long latency periods such as Alzheimer’s, ALS, or Parkinson’s, however, may prove difficult to model in relatively short-term in vitro cultures. It may be unrealistic to expect a phenotype to be revealed after a few weeks in culture of a late onset disease that takes decades to appear in patients. An additional complicating factor is that cells derived from iPS cells are more fetal in nature, reflecting a young developmental stage. External factors mimicking some type of environmental stress may be required to speed the in vitro aging process.

The true power of iPS technology is the potential to create multiple cell types from a single patient that are believed to interact in the development and progression of complex diseases. As multiple cell lineages are incorporated into disease models, researchers are exploring ways to recreate the three-dimensional in vivo setting for these models. Advances in tissue engineering will enable multiple cell types to interact and communicate with each other in a manner that more closely resembles their in vivo environment. Incorporation of advanced scaffolds and matrices may reveal phenotypes that are not obvious with single cell types or even multiple cell types co-cultured in a two-dimensional environment.

Understanding the microenvironment that iPS derivative cells may face when they are transplanted back into the body is also critical. Dr. Aileen Anderson and her team at the University of California at Irvine are exploring whether neurons derived from fetal neural stem cells, embryonic stem cells, and iPS cells can be used to mediate repair in spinal cord injuries. Their focus is to understand what the role of the inflammatory microenvironment will be in dictating how a cell population responds after transplantation. The team is studying how cells from these different populations are going to be influenced after transplantation in terms of their fate, their migration, and how the environment they see is going to signal back to those cells.

Drug screening

iPS derivative cells offer unique advantages when incorporated into the traditional small-molecule drug discovery and development process. Disease-specific iPS derivative cells can be used to assess and optimize lead compounds and facilitate metabolic profiling and toxicity evaluation.

Using iPS derivative cells, potential therapeutics can be screened against a large number of patient-specific cells prior to initiating clinical trials. Variation in the response to drugs by cells of patients with genetic differences can guide more targeted selection of patients for enrollment in clinical trials, resulting in trials that are smaller and more likely to be successful.

Dr. James Ellis, senior scientist at the Hospital for Sick Children in Toronto and co-scientific director of the Ontario Human iPS Cell Facility, is interested in drug screening applications for cystic fibrosis. He and his group are looking to identify reproducible phenotypes first. Once this is done, high-throughput screening will be a very effective way to identify small molecules that may be effective for a particular patient.

Dr. Ellis readily sees the value in the derivation of lung cells from iPS cells. According to him, “Obtaining lung cells from a patient with cystic fibrosis is really only possible when they’ve undergone a lung transplant. But one of the consequences of the disease is that patients have dramatic lung infections so it’s very difficult to establish primary cell lines and even if you did, those will have a limited ability to be passaged. You may not be able to make enough cells to complete or verify your screen.”

Through use of iPS cells, the laboratory plans to generate large numbers of cells from a range of patients. Genomic patterns can then be cross-referenced to drug screening results. The researchers can then compare one patient to others and possibly start to make predictions as to which drugs are going to work in which patients.

Investigative toxicity

A significant advantage of using iPS cells in drug screening applications is the ability to conduct toxicity tests on cells of the same patient. If a drug is found to be efficacious, it can be tested against cardiomyocytes and hepatocytes derived from the same iPS cells. Use of iPS cells for toxicity testing can also help overcome the inherent limitations of current methods.

The liver plays a central role in processing and metabolizing drugs and other substances in the bloodstream. Because hepatocytes are responsible for metabolizing most compounds that enter the body, these cells are used during the drug discovery process to predict how drugs will be metabolized and to what extent a drug may be toxic to the liver.

Drug-induced liver injury is the principal reason clinical trials are suspended and approved drugs withdrawn from the market. In fact, drug-induced liver injury has been the most frequent, single cause of safety-related withdrawals of marketed drugs over the past 50 years.6

Investigative in vitro liver toxicity studies are typically conducted using primary human hepatocytes or an immortalized (genetically transformed) liver-derived cell line such as HepG2. Despite routine use for investigative toxicity, both of these options present significant drawbacks:

  • Primary human hepatocytes are derived from fresh liver tissue, which is typically sourced from cadavers or cancer resections. Supplies can be limited and the tissue can vary widely among donors.
  • Primary hepatocytes cannot be sustained for more than a few days in culture without losing function.
  • Although immortalized hepatocyte cell lines can be cultured indefinitely, these cells display distinct differences from normal liver cells and may not exhibit normal cell behavior or response.

The challenges of hepatocyte-based in vitro toxicity testing have led drug developers to rely heavily on animal models for preclinical metabolism and toxicity testing. But animal models may not be fully and reliably predictive of human toxicity, are low throughput and expensive, and raise ethical concerns for some.

Cost and throughput often relegate use of animal models to the later stages of preclinical development, after a company has invested significant resources and time in a lead compound. This delayed evaluation of toxicity contributes to the high failure rate of compounds in late-stage preclinical testing, which is extremely costly. Earlier, more effective assessment of drug candidate toxicity has the potential to reduce the attrition rate of drugs in later stages of development.

Differentiation and expansion of human iPS cells into functional hepatocytes for use in investigative toxicity studies could overcome the shortcomings of primary hepatocytes and immortalized cell lines. Use of iPS-derived hepatocytes (and other cell types commonly used for toxicity studies) offers a number of important advantages to investigative toxicity studies, including:

  • Availability of a consistent source of cells that more closely match in vivo phenotype and physiology
  • Elimination of reliance on donor sources that can have sporadic availability
  • Reduction in the use of animal models and animal tissue
  • A more standardized, reproducible process for toxicity testing
  • Improvement in the predictive capabilities of early toxicity studies, leading to reduction in late-stage attrition of drugs.

More efficient and predictive toxicity studies enabled by iPS-derived cells can be expected to reduce development costs associated with the late-stage failure of drug candidates. Identifying drug candidates with toxicity concerns earlier in the discovery process can improve the safety and, ultimately, the successful outcomes of clinical trials.

The ability to differentiate iPS cells into a wide range of lineages has created new opportunities in both clinical and research settings. Use of these cells for disease modeling and drug and toxicity screening can help overcome the limitations of current methods, enable construction of human models of complex diseases, and reveal important insights that can lead to a more personalized approach to medicine. Alongside their potential to reshape the research and clinical landscape, these remarkable cells have presented researchers with new questions to explore, challenges to address, and applications to discover.

References

  1. Takahashi, K.; Tanabe, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factor. Cell2007, 131, 861–72.
  2. Baker, M. Integration-free iPS cells. Nature Reports Stem Cells; Oct 16, 2008.
  3. Sommer, C.A.; Gianotti Sommer, A. et al. Excision of reprogramming transgenes improves differentiation potential of iPS cells generated with a single excisable vector. Stem Cells2010, 28(1), 64–74.
  4. Jaenisch, R.; Young, R. Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell 2008, 132(4), 567–82.
  5. Warren, L.; Manos, P.D. et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell2010, 7(5),618–30. In press.
  6. Guidance for Industry: Drug-Induced Liver Injury; U.S. Department of Health and Human Services, July 2009.

Dr. Chu is R&D Manager, and Ms. Noble and Ms. Rollins are Product Managers, Stem Cells/Cell Biology, EMD Millipore, 290 Concord Rd., Billerica, MA 01821-3405, U.S.A.; tel.: 781-533-6000; e-mail: vi_chu@millipore.com.

Comments