World’s Largest hiPSC Bank: Made in California

By on April 27, 2017

Authored by Debbie King, Contributor, Cell Culture Dish

The Importance of Quality Cell Banks

The value and need to establish stem cell banks of validated and quality-controlled human pluripotent stem cell (hPSC) lines has long been recognized by researchers to ensure that work from different laboratories worldwide could be replicated and compared. These cells are used for research applications such as developmental biology, toxicology and drug discovery and to study their potential in cell therapy. These investigations are being performed with many pluripotent stem cell lines grown in a variety of culture conditions making standardization difficult and could generate cells that have acquired permanent deleterious changes. The consequence of erroneously using such cells is not only wasted time and resources but, more importantly, the generation of incorrect data that, if published, could both confuse and delay scientific progress (Stacey et al, 2013). Therefore, ensuring that cell lines used in the pluripotent stem cell field are properly validated and characterized is vital to achieving high quality research.

Both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have the capacity to generate all of the cell types in the human body making them valuable tools for investigators. In 2006, the induced pluripotent stem cell (iPSC) technology was pioneered by Shinya Yamanaka’s lab in Kyoto, Japan. He showed that the introduction of four transcription factor-encoding genes (Oct4, Sox2, cMyc, and Klf4) could return adult cells back to a pluripotent or ‘embryonic-like’ state. Because adult cells are used, they are fewer restrictions and controversy surrounding their use compared to human embryonic stem cells (hESCs), which are derived from pre-implantation stage human embryos, thus increasing demand for quality hiPSC lines. The expertise, time and cost required to derive and validate iPSC lines are a key impetus for the creation of quality iPS cell banks (O’Rouke et al, 2008).

Through the efforts of the scientific community, the generation of human embryonic stem cell banking networks has ensured that well-characterized and quality controlled hESC lines are broadly accessible to researchers worldwide. The hard-won experience achieved in producing hES cell banks for research has paved the way for the creation of hiPSC cell banks in a similar fashion for research and drug discovery (Stacey et al, 2013). The momentum of hiPSC research shows no signs of slowing down and the field would benefit greatly from cell banks that provide qualified, traceable, and reliable sources hiPSC lines to investigators worldwide. The basic guidelines for international banking and supply of pluripotent stem cells are described by the International Stem Cell Banking Initiative (Andrews et al, 2009) and include important features such as informed consent, traceability, and cell characterization.

California’s Stem Cell Initiative

The criteria mentioned above were carefully considered when the San Francisco-based California Institute for Regenerative Medicine (CIRM) embarked on The Human Pluripotent Stem Cell (hPSC) Repository project. This initiative aims to provide the world’s largest biorepository of high quality, disease-specific induced pluripotent stem cells to be used globally by non-profit and for-profit organizations. California’s Stem Cell Agency was created in 2004 when Proposition 71: the California Stem Cell Research and Cures Initiative was approved and serves as the state’s stem cell research agency with $3 billion in funding support . The hPSC Repository (with a $32 million budget) is one of CIRM’s major efforts to provide valuable resources to the global research community through partnerships with both academia and industry. Jonathan Thomas, Ph.D., J.D., CIRM Chairman, said, “We believe the bank will be an extraordinarily important resource in helping advance the use of stem cell tools for the study of diseases and finding new ways to treat them. While many stem cell efforts in the past have provided badly needed new tools for studying rare genetic diseases, this bank represents common diseases that afflict many Californians. Stem cell technology offers a critical new approach toward developing new treatments and cures for those diseases as well.”

CIRM established key partnerships with academic and industry leaders for the hPSC Repository (Figure 1). In March 2013, Wisconsin-based Cellular Dynamics International (CDI) and New Jersey-based Coriell Institute for Medical Research (Coriell) received two multi-million dollar grant awards from CIRM for this cell banking project. CDI was awarded $16 million to create 3 induced pluripotent stem cell (iPSC) lines for each of 3000 healthy and diseased donors representing eleven diseases. In conjunction, Coriell was awarded almost $10 million to establish a biorepository for these lines. The balance of the $32 million was awarded to four California institutions for recruitment of tissue donors to create the iPS cells.

Figure 1: CIRM Human Induced Pluripotent Stem Cell (hiPSC) Initiative Awards

(Figure obtained from https://www.cirm.ca.gov/about-cirm/newsroom/press-releases/03192013/stem-cell-agency-banks-32-million-new-approach-advance Accessed March 28, 2017)

Kaz Hirao, CDI Chairman and CEO, said, “iPSCs are proving to be powerful tools for disease modeling, drug discovery and the development of cell therapies, capturing human disease and individual genetic variability in ways that are not possible with other cellular models. We’ve seen a dramatic increase in the availability of iPSC lines. We’re pleased to be the vendor of choice for creating high quality iPSC lines and enabling scientists from academia and industry to better understand and help develop treatments for major diseases.”

“Coriell Institute has established expertise in iPSC technology for several years now, and this award will expand our presence in the field considerably,” said Michael Christman, Ph.D., President and CEO of Coriell. “We’ve also determined the best practices in biobanking during our 60 years of operations, managing millions of biological specimen for research discovery around the globe. We see the vast research potential with iPSCs and are enthusiastic about partnering with an outstanding company like CDI which is a world leader in the field.”

Even though both CDI and Coriell are based outside California, facilities were established at the Buck Institute in Novato, CA, where the iPSCs are generated and banked. Both companies occupy about 4,500 square feet of space at the Buck Institute’s Regenerative Medicine Research Center.

Key Features of the hPSC Repository

The size, the careful selection of diseases, its stock of high-quality cells and widespread public access differentiates this repository from similar efforts (i.e. StemBANCC and HipSci). The large size of this cell bank makes it unique for the study of genetic variation between individuals to improve our understanding of how disease and treatment varies in a diverse population that would otherwise be impossible with a smaller cell bank. Many of the common diseases represented in the collection have a complex or unknown origin that makes it difficult to develop effective treatments and would benefit from such large-scale comparisons. The hPSC Biorepository’s disease areas of focus include:

  • Cardiovascular disease
  • Lung disease
  • Liver disease
  • Blinding eye diseases
  • Childhood neurodevelopmental disorders (i.e. infantile epilepsy, autism and cerebral palsy)
  • Alzheimer ’s disease

iPSCs are unique in that they can be generated from cells obtained from adults afflicted by a known disease and then differentiated into any cell type in the body. For example, comparison of the heart cells differentiated from both diseased and healthy donor iPSC lines in the laboratory could help uncover the underlying genetic cause of the disease by recapitulating the disease biology. “The cell bank will allow researchers to test new treatments faster and more precisely before embarking on a large, expensive clinical trial”, said Joseph Wu, director of the Stanford Cardiovascular Institute and a professor at Stanford University Medical School.

The hPSC Biorepository became publically available on September 1, 2015 through Coriell’s online catalog (https://catalog.coriell.org/CIRM). As of March 2017, there are 1211 validated cell lines available to researchers worldwide.

The aim of CIRM’s hPSC Repository is to provide global access to the highest quality hiPSC lines as a valuable resource for the research community as opposed to being ‘for-profit’ (in contrast to other commercial cell banks). Therefore, the cells are being provided at cost, which will hopefully allow the bank to sustain itself while maintaining cost-effectiveness for researchers. One vial of cells costs $750 for academic or non-profit organizations and $1,500 for commercial entities.

The generation of the hPSC Repository can be broken down into three main steps: tissue collection, iPS cell line generation (CDI) and iPS line banking and distribution (Coriell).

Tissue Collection

CIRM wanted to establish guidelines early on ensuring patient understanding of how their donated cells would be used for the biorepository. This “informed consent” is the basis of research ethics and is particularly challenging for iPSC research because the cell lines generated from donor tissues are immortal and could be used for research for many years to come. At CIRM, the governing board turned to the Standards Working Group (SWG) – comprised of scientist, patient advocates, and medical ethicists – to advise them on ethical procedures for responsible research conduct (Lomak, 2012). The group noted the need to emphasize to prospective donors that their cells will not be for personalized therapies. Instead, the reprogrammed cell lines would be deposited in a cell bank to aid scientific advancement. As a result of the discussions, CIRM developed a rigorous model for informed consent encompassing many potential downstream uses for the iPSC lines prior to recruitment, including any commercialization of products that could result from use of the cells. This provides peace of mind for researchers knowing there will be minimal complications for their research programs resulting from flawed consent. Each donor’s consent included statements on the following:

  • Testing the cells’ DNA (this is referred to as the cell’s genetic code or sequence) and making the information known to other researchers
  • Changing some of the genetic code or sequence within these cells
  • Using cells to test or select drugs to treat disease
  • Transplanting cells or resulting products to humans or animals
  • Distributing cells widely (nationally and internationally) for research, training or commercial medical product development
  • Future research and uses unforeseen at this time

Once they have consented to participation, the patients are screened and negative for communicable diseases such as HIV, HBV, and HCV. Peripheral blood mononuclear cells (PBMCs), obtained via blood draw, and skin biopsies were procured from patients recruited by the tissue collectors listed in Figure 1 and deposited with the biorepository operated by Coriell. However, due to delays in obtaining sufficient donor samples, possibly resulting from the tight timeline for the project, a one-year extension was approved to complete the activities associated with the grant by 30 November 2017.

iPSC Line Derivation: Cellular Dynamics International

Cellular Dynamics International (CDI), a FUJIFILM company, is a world leader for the manufacturer of human cells used in drug discovery, toxicity testing, stem cell banking, and cell therapy development. Their expertise in generation of iPSC lines was an asset to the CIRM cell banking initiative. After the primary tissues were cataloged by Coriell, they were given to CDI for iPSC line derivation. CIRM ensured consistency in cell line generation for the cell bank by having one entity deriving all the lines unlike other hPSC banks where lines are generated and deposited by different investigators (with varying derivation, culture and cryopreservation techniques). Having uniformity in derivation and culture methods allows the hPSC repository cell lines to be directed compared because the cells share the same ‘history’.

The donor cells were reprogrammed using an episomal method – pioneered by Junying Yu and James Thomson (Yu et al, 2009) -where circular DNA vectors deliver the reprogramming genes into the donor’s somatic cells to generate the iPSC lines. Episomal reprogramming is superior to other reprogramming methods, especially for clinical applications, because it is non-integrating (i.e. vector DNA will not incorporate into host genome). The large number of iPSC lines for the hPSC Repository necessitated a heavy reliance on automation to conduct the high-throughput iPSC reprogramming. By the end of 2016, iPSC clones from 1737 donor samples had been generated for the initiative.

The iPSC lines were derived and processed in a standardized manner to ensure quality standards were maintained. They were cultured in feeder-independent conditions using Essential 8 (E8) culture media on a vitronectin matrix and cryopreserved in colony form. Additionally, CDI used their established quality control pipeline to guarantee each cell lines’ pluripotency and genetically stability. Figure 2 summarizes the rigorous quality testing criteria each line must pass.

Figure 2: Quality Control Criteria for iPSC Lines

(Figure obtained from https://catalog.coriell.org/1/CIRM/About/cirm-lines Accessed April 1, 2017)

Each iPSC line was carried to passage 10 for deposit at the Coriell Institute, where they are stored and distributed globally to academic and industry researchers.

iPSC Banking and Distribution: Coriell Institute

The Corielle Institute has years of expertise managing complex biospecimen collections (from tracking and safe storage to distribution), but were also challenged by the sheer volume of samples and data in this collection. There are approximately 40 vials for distribution from each of the 3000 main iPSC lines requiring cryostorage, not including the primary donor tissues (i.e. PBMCs and skin biopsies) also requiring proper storage and tracking. Therefore, a Clinical Information Management System (CIMS) was established to help track all the information associated with the cell lines. In addition to providing traceability for the iPSC lines generated by CDI, Coriell also collected the de-identified clinical data for each patient (such as patient gender and whether an individual has a specific disease). All of this data is available to researchers through Coriell’s on-line catalog for every iPSC line.

The Future of Stem Cell Banks

With continued advances in using iPSCs for disease modeling and drug screening, the hPSC Repository’s size and diversity will facilitate researchers in identifying the underlying genetic causes of specific diseases with hopes of leading to novel treatment options. CIRM was able to leverage the expertise from CDI and Coriell to establish a robust framework for population the cell bank with authenticated cell lines of the highest quality. Having access to a reliable source of cells will streamline and accelerate research by allowing investigators to focus on achieving their research goals by using the cells themselves rather than spending money and time on their derivation and validation. Stem cell banks will increasingly becoming an industrial-scale activity rather than an informal exchange network among individual labs as the number of researchers working on stem cells increases and new applications are investigated. They will also need to grow and adapt to the rapid changes in the dynamic field of stem cell research, in particular, for regenerative medicine applications.

The potential of iPSCs for cell therapies in regenerative medicine remains a hot topic. But, despite what is often reported by the media, researchers have recognized that personalized or autologous therapies may not be feasible for treatment of disease because of the wait time and high cost to derive iPS lines for each patient. For example, the first ever in-human clinical trial conducted by Kobe-based RIKEN Center for Developmental Biology, led by Dr. Masayo Takahashi using retinal pigment epithelial (RPE) cells for treatment of exudative (wet-type) macular degeneration (AMD) cost roughly $1 million US dollars and one year to complete.

Undoubtedly, autologous cell therapy would be ideal because there would likely be no immune suppression post- transplantation but recent changes in the clinical landscape by some key researchers could suggest a substantial shift in the field toward an allogeneic (where donors and recipients are genetically similar but not identical) approach to disease treatment. While there is the potential for transplant rejection with allogeneic donors, the major advantages of mass-production and standardization may offset this. Japan in particular, is at the forefront of the field having moved very quickly to human clinical trials for a number of diseases. Dr. Masayo Takahashi announced that the second patient in the Japanese AMD clinical trial (mentioned above) would be treated using allogeneic cells from an established iPS cell bank that have been extensively profiled. A separate clinical trial set to come online for treatment of Parkinson’s disease, led by Dr. Jun Takahashi, will follow suit. The switch from autologous to allogeneic therapy could be an important one when looking ahead to global clinical therapy (Knoepfler, 2015).

For now, iPSC lines for clinical use would benefit from inclusion of a broad range of human leukocyte antigen (HLA) genotypes to cover a wide population for allogeneic treatments. HLAs allow the immune system to distinguish “self” from “non-self” and are a key part tissue and organ transplants. HLA superdonors are individuals whose genetic HLA profiles make their cells more compatible for donation to unrelated patients. Already, companies such as Lonza (Basel, Switzerland) and Cellular Dynamics are generating and banking their own HLA superdonor cell lines using good manufacturing practices (GMP) for therapeutic purposes. Currently, CDI has two HLA superdonor cell lines, providing a partial HLA match to 19% of the U.S. population and plans to continue adding to the bank that will match 95% of the population. Taken together, the need for continued efforts from CIRM, CDI and other organizations to fund large-scale cell banking efforts (whether for research or clinical pursuits) remains an important objective to accommodate the ever-changing scientific landscape.

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