Stem Cells and Toxicity Testing: An Update, Part I

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In the Spotlight

Stem Cells and Toxicity Testing: An Update, Part I

Sherry Ward, AltTox Contributing Editor

Published: April 4, 2013

(This is Part I of a three-part series; see also Part II and Part III.)

Stem cells – with their unique ability to regenerate themselves and the different tissues of the body – have been known to exist for decades. However, it is only recently that scientific discoveries and technological innovations have emerged that allow us to pursue the use of stem cells in novel approaches that address key scientific and medical challenges (see table “Stem Cell Milestones”).

Seven new cell lines were added to the National Institutes of Health (NIH) Human Embryonic Stem Cell Registry in March 2013, making a total of 207 human embryonic stem cell (hESC) lines available for use in NIH grant-funded research. In November 2012, a new human pluripotent stem cell database, StemCellDB, was also announced by the NIH. StemCellDB is a gene expression search engine for undifferentiated (hESC) and differentiated cells.  Research at the NIH to establish StemCellDB set a baseline for pluripotent stem cells – a list of the genes that define pluripotency.

Stem Cell Milestones*

Year

Breakthrough

Who

1962 Frog was cloned John B. Gurdon
1981 Mouse embryonic stem cells: stem cells were isolated from early mouse embryos Gail R. Martin; M. J. Evans & M. H. Kaufman
1992 Neural stem cells were identified in adult human brain
1995 Isolation of primate embryonic stem cell line James Thomson, et al.
1998 Human embryonic stem cells: stem cells were isolated from early human embryos, and human embryonic stem cell line grown in the laboratory James Thomson, et al.
2001 Dermal stem cells were identified in adult skin
2006 Mouse induced pluripotent stem cells: reprogrammed adult mouse skin cells that have embryonic stem cell characteristics were developed Shinya Yamanaka
2007 Human induced pluripotent stem cells: reprogrammed human skin cells that have embryonic stem cell characteristics were developed Shinya Yamanaka, et al.; Junying Yu, et al.
2009 iPSCs created without use of retroviruses Andras Nagy
2010 Adult cells were reprogrammed directly into neurons, cardiac, muscle, and blood cells

*A detailed Stem Cell Timeline has been published elsewhere. Only a few of the noteworthy “firsts” are listed here.

The use of stem cells in toxicity testing is relatively new, with the most recognized application being the embryonic stem cell test (EST), which was developed in 1997 for embryotoxicity testing. A search of the PubMed database on March 20, 2013 showed a total of 1448 citations to publications identified in a search for “stem cells AND toxicity testing,” beginning with one in 1970 and not increasing substantially until around 2001. By 2010-2012, well over 100 papers were cited per year.

For those unfamiliar with stem cell terminology, there are many resources available, such as the NIH’s Stem Cell Basics, EuroStemCell, and Canada’s Stem Cell Network. A brief review of stem cells and stem cell terminology, based primarily on information from Stem Cell Basics, will be provided here before discussing some of the current uses of stem cells in toxicity testing (in the upcoming Part II of this article).

Stem Cell Primer

Stem cells are the precursors of all other cells in the body. Stem cells divide originally to create the embryo and later replicate in an organism when needed to regenerate and/or repair tissues. Stem cells are unspecialized (undifferentiated) cells that when the cell divides (either symmetrically or asymmetrically) can remain as undifferentiated stem cells or can differentiate into the cells that form a specific tissue or organ.

The terminally differentiated cells that comprise most of the cells in the body do not have this capacity of stem cells to form any cell type; these differentiated cells can only produce cells of their own type when they divide.

Stem cells can be categorized according to their lineage potential as:

  • Totipotent: cells of the very early embryo (first few cell divisions) that are capable of forming the entire organism
  • Pluripotent: cells that can differentiate into all the different cells of an organism, except for placenta and amniotic sac (not sufficient to form the full organism)
  • Multipotent: cells that can differentiate into more than one type of non-stem cell, but within a related group of cell types
  • Unipotent: cells that can differentiate into only one type of non-stem cell

The three types of stem cells are embryonic, somatic/adult, and induced pluripotent.

Embryonic stem cells (ESCs) are undifferentiated, pluripotent stem cells that are derived from 3 to 5 day-old embryos (inner cells of the blastocyst stage before formation of the tissue germ layers). ESCs are capable of dividing and proliferating for a year or longer in the laboratory, while remaining in their undifferentiated stem cell state. This fundamental property of stem cells is called long-term self-renewal. Certain cell culture conditions and environmental factors are required to propagate stem cells while maintaining them in this undifferentiated/unspecialized state. “It has taken scientists many years of trial and error to learn to derive and maintain stem cells in the laboratory without them spontaneously differentiating into specific cell types.”

Embryonic stem cells are thought to be the most versatile cells for research, because they can differentiate into any type of cell in the body, i.e., they are pluripotent. ESCs are also easy to propagate in cell culture, so can provide the large numbers of cells needed for research. Their application in regenerative medicine (human therapeutics) might be limited due to the potential for immune rejection, because the cells originated from an unrelated donor (i.e., the embryo). Techniques for achieving the complete and reproducible differentiation of ESCs to specific cell lineages are still being investigated and developed.

Somatic or adult stem cells are undifferentiated cells residing in various tissues of the body that retain the capability of repopulating/repairing a tissue when needed. An example of an adult stem cell population would be the corneal epithelial stem cells located in basal cell layers of limbal region of the cornea. These limbal stem cells are thought to be the source of new corneal epithelial cells, which migrate to the surface of the cornea as they replace older cells or repair epithelial injuries. Specific areas of a tissue that contain adult stem cells are called stem cell niches.

Somatic/adult stem cells have the main limitation of being able to differentiate only into the cell type of their original tissue/organ. However, there have been reports of somatic stem cells differentiating into other cell types, a process called transdifferentiation. If transdifferentiation does occur, the process seems to be inefficient and it is uncertain that it can occur in human cells. Somatic stem cells are present in only very small numbers in the various tissues and organs, and are generally difficult to isolate and identify. Their capacity to divide in culture is also limited, so they are not available in the quantities needed for therapeutic or research applications. However, there are some important potential applications for the use of adult/somatic stem cells. Since a patient’s own stem cells could be multiplied, modified, and used as a therapeutic, they would be less likely to be rejected by the patient’s immune system. Furthermore, the cells could provide unique disease-specific models for research and drug testing.

A special type of adult stem cell often mentioned in the literature is the mesenchymal stem cell (MSC). MSCs are a heterogenous group of pluripotent cells most commonly isolated from the bone marrow, but have also been found in up to 30 other organs/tissues, which can differentiate into mesenchymal tissues (osteoblasts, chondrocytes, and adipocytes). MSCs have been found to have some important clinical applications, but their name, definition, and stem cell potential remain controversial.

Induced pluripotent stem cells (iPSCs) are reprogrammed cells that become pluripotent, like embryonic stem cells, due to genetic manipulation to induce expression of or introduce certain embryonic genes. In 2006, Shinya Yamanaka added four genes to adult mouse skin cells and within several weeks the previously differentiated cells were “reprogrammed” into pluripotent stem cells. Although iPSCs behave like embryonic stem cells, possible differences are still being explored. Incomplete reprogramming and/or genetic instability in iPSCs have been reported as possible causes for genetic differences between ESCs and iPSCs. Techniques for achieving the complete and reproducible differentiation of iPSCs to specific cell lineages are active areas of research.

New techniques for making iPSCs (non-viral gene delivery) have made them potentially safer for use in transplantation, leading to the current surge in research to develop patient-specific cell-based treatments for diseases such as diabetes and Parkinson’s. Cultured human pluripotent stem cells (hPSCs or hiPSCs) are also being used as disease models for research and for testing the efficacy of new drugs, studies sometimes referred to as “disease in a culture dish.” hiPSCs are now providing researchers with unlimited amounts of stem cells for research and potential clinical applications that do not have the ethical stigma or limited availability encountered when using human embryonic stem cells.

Whether iPSCs or ESCs are being used, the directed differentiation to induce the pluripotent cells to become a particular cell type is important to the ability to use the cells in research or as therapeutics. The differentiation of stem cells is controlled by a variety of signals, and occurs in several stages. Each stage of differentiation is triggered by internal (genes) and external (molecules in the microenvironment and physical contact with other cells) signals. Identifying the signals that induce differentiation to specific cell types is another important area of current investigation.

Stem Cells in Models for Toxicity Testing

The most attention-grabbing stem cell breakthroughs covered in science news involve the promise of their uses in regenerative medicine (cell and tissue transplantation), as pre-clinical models for drug development, and as “disease in a dish” models for drug efficacy and mechanistic studies (2012, 2013).

However, stem cell technologies have also led to many new developments in the field of toxicity testing. Human stem cell-derived in vitro models hold great potential for the development of biologically relevant models for evaluating the toxicity of substances to humans.

In Part II of Stem Cells and Toxicity Testing: An Update, some of the current applications of stem cells in models used for toxicity testing will be reviewed, including stem cell models for reproductive toxicity testing, pre-clinical testing models, clinical trials in a dish, and bioengineered 3-D human tissue models. Recent progress at incorporating stem cell niches into a bioengineered human tissue model is perhaps the most interesting development that will be highlighted in Part II.

“The Promise of Stem Cell Research,” Terese Winslow, © 2008.

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