Drug toxicity that emerges either in late-stage clinical trials or following market launch has been a long-term problem for
the pharmaceutical industry. The fundamential challenge is that existing preclinical models do not adequately predict the
toxicity of new chemical entities. Current cell models are either primary cell cultures derived from non-human animals or
immortal cell lines derived from tumors. Because of their non-human nature or neoplastic life history, they are imperfect
predictors of drug toxicity in humans. Recent advances in stem-cell technology hold the potential to overcome such limitations.
CELLULAR DYNAMICS INTERNATIONAL
The identification and isolation of stem cells from human embryos opened the promise of using human cells to more efficiently
drive drug discovery and toxicity assessments (1). At some point, stem cells may also be used in regenerative medicine to
repopulate or even replace diseased tissues and organs. Human embryonic stem cells (ESCs) can differentiate into any of the
200-plus cell types found in the human body.Unfortunately, human ESCs have their drawbacks. Because of their source, ethical
controversy has surrounded ESC research since the cells were first isolated in 1998. Furthermore, as they are derived from
surplus embryos from in vitro fertilization procedures, their sources and genetic make-up are unknown, thus limiting their utility for investigating potential
compound toxicity in targeted human subpopulations.
Induced pluripotent stem cells (iPSCs), on the other hand, possess the advantages of ESCs, avoid the associated ethical implications,
and have the intrinsic ability to be a more flexible cell-based research tool. iPSCs are produced from somatic cells that
are induced to a pluripotent state by the incorporation of genetic and small-molecule factors that reprogram the somatic cell
to a stem cell (2–4). iPSCs have the same pluripotent capabilities of ESCs and the advantages of originating from individuals
with identifiable phenotypes and genotypes, thus enabling the use of targeted human subpopulation models early in drug discovery
and toxicity screening.
Producing cardiomyocytes from human ESCs and iPSCs using the embryoid body (EB) and directed differentiation methods is well
documented (5, 6). The efficiency with which these methods can differentiate ESCs and IPSCs into cardiomyocytes is highly
variable, but consistent among both methods is a difficulty in producing highly pure (> 90%) populations of cardiomyocytes.
Additional comparisons of cardiomyocyte generation from human ESCs and iPSCs suggest that both starting materials have an
equivalent cardiogenic capacity (7), and further molecular and electrophysiological analyses demonstrate that both starting
materials produce differentiated cardiomyocytes with phenotypes consistent with atrial, ventricular, and nodal cardiomyocyte
subtypes (5, 6).
As described above, human iPSCs have particular advantages over ESCs for drug discovery and toxicity testing when induced
to differentiate into a mature cell type. The significant challenge for commercialization of such cells is the ability to
consistently produce both starting material iPSCs and the differentiated cells in the quantity, quality, and purity required
by the pharmaceutical industry. Here we will describe a process by which we have industrialized the manufacture of iPSC-derived
human cardiomyocytes. This process, as currently practiced at Cellular Dynamics International (CDI), is capable of meeting
the foreseeable demand for purified iPSC-derived human cardiomyocytes and is scalable by more than two orders of magnitude
if necessary without difficulty. We will illustrate the purification process and show that the human cardiomyocytes generated
from this process express the same genes, proteins, electrophysiological properties, and responses to cytotoxic substances
and ion channel blockers expected of cardiomyocytes.
Manufacture of human iPSCs
iPSCs are developed from human fibroblasts, skin, blood, or other somatic cell types collected from individuals. The purified
somatic cells are exposed to reprogramming agents that stimulate de-differentiation into iPSCs. Typically, reprogramming somatic
cells is a slow and complicated process to which many investigators have contributed different methodologies (8). Originally,
the reprogramming process required genetic modification of the source material with viral vectors that permanently integrate
into seemingly random locations within the host DNA. Although the reprogrammed cells exhibited the properties of stem cells,
the integrated vectors limited the usefulness of the cells to clinical or drug testing applications because of the perceived
potential mutagenic or oncogenic effects of the integrated DNA. New reprogramming methodologies have been developed that largely
overcome this barrier (9, 10) so that reprogrammed cells do not have foreign DNA, vector or otherwise, integrated into the
genome, thus creating iPSC lines with potential applications in clinical as well as discovery settings.
The key to the mass production of iPSCs is to develop a process that is both scalable and standardizable (see Figure 1). iPSCs,
by their nature, are highly proliferative and have the potential to greatly expand their numbers under cell culture conditions.
However, they are also very sensitive to manipulation and thus require special treatment and expertise to prevent entry into
various non-directed differentiation pathways, events which greatly reduce the ability of the cell population to undergo directed
differentiation and reduce the health of the relatively few terminally differentiated cells that are produced.
Figure 1 ALL FIGURES ARE COURTESY OF CELLULAR DYNAMICS INTERNATIONAL (CDI)
To prevent entry of iPSC populations cultured under standard conditions into non-directed differentiation pathways, such differentiated
cells must be removed and "weeded out" of the population. This weeding step is subjective, labor-intensive, and highly dependent
on the skill and attention of the technician. Therefore, to manufacture the necessary number of iPSCs needed to produce terminally
differentiated cells for use by the pharmaceutical industry, we have simplified the process to enable standardization and
assembled it into a highly parallel structure.
The major production constraint of cell weeding was eliminated by developing a proprietary culture system that a) used standard
single-cell splitting techniques to eliminate the need for periodic weeding, and b) added small molecules to the cell cultures
to promote survival and proliferation
Scalability was incorporated into the process by building the cell culture system in a highly parallel nature that enabled
the production of billions of iPSCs through the use of CellSTACK culture chambers (Corning, Lowell, MA) rather than standard
tissue culture or T-flasks. The large surface area to footprint ratio of the CellSTACK system enabled parallel culturing of
iPSCs and resulted in a significant expansion in iPSC production. Currently, this "industrialized" process can generate 100
billion or more iPSCs per month with a small team of manufacturing technicians. Because manufacturing is standardized, production
levels can be increased through the addition of additional cell-culture manufacturing lines.