Introduction


Conceptually, stem cell research can be viewed as a branch of modern biology that attempts to create stem cells from differentiated cells or to transform embryonic or adult stem cells into specialized, differentiated cells that can be used to replace damaged cells or organs. Research conducted from 1998 to 2015 on human stem cells has demonstrated that the transformation of stem cells into healthy specialized cell types is emerging as a fundamental biological area of study that could lead to revolutionary therapies and clinical applications. Many scientists are convinced that stem cell research also will lead to a better understanding of fundamental aspects of biology in the areas of cellular differentiation, organ regeneration, regenerative medicine, and epigenetics as well as the science of cancer. In this light, stem cell research simultaneously represents a domain of both critical basic research and promising clinical application. In sum, stem cell research is rapidly advancing science in profound ways, and has great potential to positively affect our health as well as our quality of life.

What are stem cells?


To more fully understand the complexities that underlie stem cell biology, it is critical to appreciate the definition of terms, understanding of the embryology, and the process of generating stem cells.

ZygoteToBlastula.png
Figure 1: Diagam showing how stem cells are formed.

Soon after fertilization, the haploid egg and sperm nuclei merge to form a single nucleus with the diploid number of chromosomes. The one-cell zygote divides as it moves along in the fallopian tube, where it continues to divide. Up until the 8-cell stage, each cell is totipotent.

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Totipotency and pluripotency


What does totipotent mean?
Soon after fertilization, the haploid egg and sperm nuclei merge to form a single nucleus with the diploid number of chromosomes. The one-cell zygote divides as it moves along in the fallopian tube, where it continues to divide. Up until the 8-cell stage, each cell is totipotent.

Totipotent means that each cell can give rise to all the 220 cell types in the embryo plus the extra-embryonic tissues necessary to form the placenta and yolk sac that together allow for the development of the fetus. The ability to form the placenta is a defining feature of totipotent cells.


Soon after fertilization, the haploid egg and sperm nuclei merge to form a single nucleus with the diploid number of chromosomes. The one-cell zygote divides as it moves along in the fallopian tube, where it continues to divide. Up until the 8-cell stage, each cell is totipotent.
As the embryo travels along the oviduct, the cells continue to proliferate and the morula develops into a blastocyst that contains a cavity. The outer layer of cells of the blastocyst will go on to form the placenta and other supporting tissues needed for fetal development in the uterus.

The inner cell mass of cells located at the polarized end of the cavity contain the embryonic stem cells. These cells are of particular interest to researchers and others as they will eventually mature to form virtually all of the tissues in the human body.




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Closed blastocyst (left) and open blastocyst (right). (Images courtesy Wellcome Library.)


These are images of blastocysts, caught on the head of a pin. In the picture on the right, the blastocyst is opened revealing the inner cell mass containing the stem cells.

What does pluripotent mean?
What is important to know here is that while the inner cell mass cells can form virtually every type of cell found in the body, and therefore the cells are considered pluripotent, they cannot form an entire organism because they are unable to give rise to the placenta and other tissues necessary for gestational development in the uterus. This is a key point. Because their potential is not total, they are not totipotent – only totipotent cells can go on to develop into a fetus. Pluripotent cells will form every cell in the body but will never form an embryo.

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Generating embryonic stem cells in vitro


We have just described the process in vivo, as it occurs in a living human female. The same process can occur in a Petri dish. An ovum (egg) and sperm can form a zygote, that becomes a morula, and continues to form a blastocyst. The scientific challenge is to isolate the pluripotent stem cells, derived from the inner cell mass of the blastula, and to stimulate these cells to undergo specialization (cell differentiation) into a cell committed to a particular function.


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Figure 2: Pluripotent, embryonic stem cells originate as inner mass cells within a blastocyst. (Source: Wikimedia Commons)

The hope is that these stem cells will be able to be harnessed for regenerative medicine: as blood cells to cure patients with leukemia, nerve cells to treat spinal cord injuries and Parkinson's disease, heart cells to replace cardiac muscle damaged after a heart attack, and pancreatic islet cells to cure diabetes. These stem cells will also provide a new laboratory model to promote genetic research and drug development - potentially substituting or, at the very least, augmenting the work already done with animal models. In summary, stem cell research has the potential for significant and diverse applications, including curative therapies.

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Early historical origins of stem cell biology

Robert Hooke (1635-1703)

Robert Hooke (1635-1703)

Robert Hooke, an Englishman, published Micrographia in 1665, which contained the first usage of the word "cell" to describe the basic unit of life. Using an early microscope of his own design and viewing a thin cutting of cork, Hooke observed empty spaces contained by walls that he termed cells. In the 1600s, the term cell suggested a small room in a monastery. Currently Hooke’s microscope is on exhibit at the National Museum of Health and Medicine in Washington, DC.
(No authenticated portrait of Hooke exists; this illustration was reconstructed from extant descriptive sources by painter Rita Greer.)


Cells as the basic units of life
The basis of stem cell biology begins with the understanding that cells form the basic units of life. In the 1600s, using his microscope, Robert Hooke observed small living compartments within cork plants. Likening the little units of cork tissue to miniature rooms or chambers, he coined the term "cells” from the Latin word cella meaning a small room.

It took the scientific community two centuries to appreciate Hooke’s initial observations. By the mid 1800s, scientists such as Theodore Schwann began formulating the cellular theory of life which contained two major conclusions:
  1. Cells are the basic building blocks of life
  2. All cells arise from pre-existing cells. Schwann’s understanding that cells have the capacity to differentiate is a key milestone in the history of stem cell research that centuries later became foundational for stem cell research.

Theodor Schwann (1810-1882)

Theodor Schwann (1810-1882)


Theodor Schwann was a German physiologist. His numerous contributions to biology include: the development of cell theory; the discovery and study of Schwann cells in the peripheral nervous system, pepsin, the nature of yeast; and the conceptualization and coining of the term metabolism.






In 1908, at the Congress of the Hematologic Society in Berlin, Russian histologist Alexander Maksimov first proposed the term “stem cell” perhaps after noting that the stem of a tree gives rise to a variety of branches.


Alexander Maksimov


Dr. Alexander Maksimov, or Maximow, (1874-1928) was a professor of histology and embryology in St. Petersburg, Russia, in the early part of the 20th century, and later a professor of anatomy at the University of Chicago. He wrote the most important book on the known histology of that period. Hematopoiesis, the theory that explains our modern concept of blood cells' origin and differentiation, is based on his work.




Cellular differentiation


As we have just illustrated, most complex organisms originate from a fertilized egg that divides, grows, and differentiates into all the various specialized cells—such as neurons, muscle cells, pancreatic cells, and blood cells—that an organism needs to function. Cellular differentiation begins with the zygote as it divides and grows. At each stage of cellular division, the daughter cells begin to transform or differentiate. Cell differentiation is a process that regulates the functional and structural specialization of cells in all organ systems within a multi-cellular organism. Specifically, cellular differentiation is controlled by a variety of parameters including local environment and the expression of specific genes, transcription factors, and proteins. Each specialized cell type turns on or turns off selected genes unique to that cell type (see Module 3 for more information about cell differentiation).

Cell specialization, for the 220 histologically different cell types characterized in the human body, is thus determined by the activation and suppression of a specific subset of the 20,000-25,000 genes representing 5% of the human genome. In addition, we are learning more about the role of the other 95% of the genome that has historically been referred to as “junk DNA,” which might not be junk after all (see Module 3 - Cellular differentiation to understand the newly discovered critical functions of “junk” DNA). (Wang, Huang et al.)

Fundamental characteristics of pluripotent stem cells


All pluripotent stem cells, are viewed by scientists as expressing two fundamental properties -- self renewal and plasticity.

Self renewal is the ability of stem cells to divide indefinitely, producing a population of identical offspring. The concept of self-renewing stem cells originated in the 1960s with McCulloch and Till who demonstrated the presence of self-renewing cells in mouse bone marrow, which we now know are hematopoietic stem cells (Becker, Mc et al. 1963; Siminovitch, McCulloch et al. 1963). Today, cell surface markers and the expression of transcription factors are important characteristics of cellular differentiation.

James Till and Ernest McCulloch


In the early 1960s, James Till and Ernest McCulloch began a series of experiments that involved injecting bone marrow cells into irradiated mice. Visible nodules were observed in the spleens of the mice that were in proportion to the number of bone marrow cells injected. Till and McCulloch called the nodules 'spleen colonies', and speculated that each nodule arose from a single marrow cell: perhaps a stem cell.




Plasticity describes the capacity of stem cells to undergo an asymmetric division, cued by environmental conditions and genetic factors, to produce two dissimilar daughter cells. As of 2015, there is still controversy whether stem cells undergo symmetical or asymmetical division. In asymmetrical division, one daughter cell, identical to the parent,continues to contribute to the original stem cell line, while the other daughter cell differentiates into specialized cell types. Symmetrical division gives rises to two identical daughter cells that are either stem cells or cells that have begun to differentiate. Plasticity also describes the ability of an organism to change its phenotype in response to changes in the environment.

But not all stem cells exhibit these properties of self renewal and plasticity. While hematopoietic and embryonic stem cells exhibit these properties, other adult stem cells may only be committed to exhibit plasticity in their ability to differentiate into other types of cells.

The hallmark property of stem cells is their ability to differentiate into a wide variety of different cell types. Thus, scientists must demonstrate that the cells they have obtained are bona fide stem cells based on their capacity to differentiate into several other types of terminal or lineage progenitor cells.

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Classification and sources of stem cells


Classification of stem cells
Classification of stem cells [see: Smith 2001 for a review on how to classify specific types of stem cells]. Stem cells can be classified into three broad categories (totipotent, pluripotent and multipotent), based on their differentiation potential. As mentioned earlier, totipotent stem cells are found only in early embryos. Each totipotent cell can form a complete organism (e.g., a baby).

Pluripotent stem cells are found in the inner cell mass of the blastocyst and have the capacity to form any of the three germ layers that compose over 200 different cell types found in the body, excluding the placenta. Multipotent stem cells are derived from adult tissue, such as umbilical cord blood and bone marrow, and generally do not have the same capacity to differentiate into all the different cell types of the human body.
Sources of stem cells
Traditionally, there have been four primary tissue sources to obtain human stem cells: embryo, fetus, neonatal (including cord blood), and adult tissue. While most tissues and organs of the human body contain stem cells, their frequency varies from organ to organ. In circulating blood, for example, only 1:100,000 cells are stem cells, while the percentage of stem cells in bone marrow is much greater.

In addition, in the adult, most organs have a unique type of stem cell that can be identified by the specific cell surface markers it expresses.

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Isolation and purification of human embryonic stem cells (hESC)


One of the most significant historical breakthroughs in human stem cell research occurred in 1998 when researchers at the University of Wisconsin at Madison, led by James Thomson, isolated and grew stem cells derived from human embryos (Thomson, Itskovitz-Eldor et al. 1998).

At the same time that Thomson reported his results, researchers from Johns Hopkins University, led by John Gearhart, described a method to isolate and culture immature germ cells from 5 to 8 week-old fetuses that were donated anonymously by women undergoing therapeutic or spontaneous abortions (Shamblott, Axelman et al. 1998). Dr. Gearhart and colleagues collected stem cells from the germinal centers of the ovaries or testes of the fetus and placed them in plastic dishes. They then added factors that enabled the germ stem cells to continue to divide, while simultaneously retaining them in a state of suspended development that prevented them from differentiating. These germ cell-derived stem cells could also be frozen, recovered, and maintained as stem cells in culture. Interestingly, Gearhart’s initial purpose for his research was merely to develop a tool for studying Down’s syndrome.


John D. Gearhart

John D. Gearhart

John D. Gearhart, PhD, Director of the Institute of Regenerative Medicine at the University of Pennsylvania, and renowned stem cell scientist, studies the molecular and cellular basis of human embryonic development. In 1998 he and colleagues published the first report on derivation of pluripotent stem cells from germ cells of the human embryo. Dr. Gearhart was a founding member of the International Society for Stem Cell Research. Source: Institute for Regenerative Medicine, University of Pennsylvania (Penn-RIM 2007) and Academy of Achievement, A Museum of Living History (Gearhart 2008).

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Induced pluripotent stem cell system (iPS) technology


In 2006 and 2007, Yamanaka and colleagues discovered that transfecting differentiated adult cells with four genes, Oct4, Sox2, Klf4, and c-Myc, could de-differentiate these cells into a pluripotent state. This process has been termed induced Pluripotent State or iPS and has generated both excitement and controversy.The excitement is based on the fact that almost any terminally differentiated cell can be coaxed to convert back into a stem cell. One scientific challenge concerning this process is that one of the genes is an oncogene (myc) that has the potential to convert a normal cell into a cancerous-like cell. While more recent efforts, fortunately, have successfully eliminated the need to use oncogenes for iPS generation, injection of iPS cells into mice still can cause tumors.

Shinya Yamanaka Shinya YamanakaShinya Yamanaka, MD, PhD, of the Gladstone Institute of Cardiovascular Disease (GICD) in San Francisco and Kyoto University, discovered a method of reprogramming adult skin cells to become embryonic-like stem cells. This process to generate stem cells was called induced Pluripotent State, or iPS. Yamanaka's discovery was made in 2006 and has created significant momentum in the field of stem cell research.


The great advantage of deriving stem cells via iPS is that this remarkable technology does not require the destruction of human embryos. Moreover, the potential of iPS means that future stem cell therapies could be based on a patient's own cells (Takahashi and Yamanaka 2006). This is a key point since the use of one’s own cells in stem cell therapy would eliminate the issue of tissue rejection, which is a critical problem in most organ donation scenarios. Tissue rejection would likely be an issue if patients were to receive stem cells from someone else. [insert religious views on the destruction of human embryos]

Thought question


What impact did the political environment during the Bush Administration have on the development of iPS? (Loike and Fischbach 2009)




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Advantages and disadvantages of stem cells from different sources


Advantages of using embryonic stem cells
One major medical advantage of embryonic stem cells is their potential to differentiate into any type of cell. Additionally, they express strong self-renewal capacity and can be maintained as stem cells in culture. Although embryos obtained from fertility clinics can provide a major source for stem cells, it is not easy to obtain these embryos and significant ethical issues are raised when the stem cells are derived from the donated embryos.

Disadvantages of using embryonic stem cells
The major disadvantages of embryonic stem cells, apart from ethical considerations, are that they may be rejected if transplanted into an HLA incompatible person, and more importantly, that they may form tumors more easily than adult-derived stem cells.

Advantages of using adult stem cells
Most adult tissues contain multipotent stem cells. The most common source for multipotent stem cells is bone marrow. Bone marrow-derived stem cells in large measure generate the multiple cell types cells found in the blood. However, scientists can direct the differentiation process of bone marrow to differentiate into a variety of other cell types (Choi, Kurtz et al. 2011). Thus, there are considerable efforts undertaken to expand the ability of adult stem cells to differentiate into even more kinds of specialized cell types.

In addition, the ease with which bone marrow cells can be obtained, coupled with our experience using these cells in a variety of treatments (e.g., leukemia), have been a great impetus for further investigation of bone marrow as a source for adult stem cells.

While bone marrow-derived cells can differentiate into a variety of blood cells and other cell types, they are not as pluripotent as are embryonic stem cells. Nonetheless, there is a significant advantage to using bone marrow or any adult-derived stem cells in autologous therapy, as the risk of tissue rejection is avoided by using the patient’s own cells.

Disadvantages of using adult stem cells
Adult derived stem cells, however, have some disadvantages in therapeutic applications. To date, disadvantages of adult stem cells are that they are:
  • few in number
  • difficult to isolate and maintain in culture
  • slow to proliferate
  • difficult to stimulate to differentiate into various other tissues types

Advantages of using iPS cells
  • obtaining iPS stem cells avoids the ethical quandaries surrounding embryo destruction
  • these cells can be derived from autologous tissue avoiding the risk of immune rejection
  • unlimited resource capacity because they require only a tissue biopsy for derivation
  • safer than obtaining embryos via IVF technology

Disadvantages of using iPS cells
  • not all iPS cells generated to date have demonstrated longitudinal functional equivalence to hES cells because of the lack of long-term follow-up studies.
  • risk of tumors.
  • iPS cells may still retain the epigenetic fingerprint of the cell type from which they were derived.

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Conclusion


From 2011 until 2015 the number of government-approved embryonic stem cell lines increased from 93 to over 300 (NIH.gov). Until we are able to test stem cells from various sources (embryonic, fetal, adult, and iPS) side by side in the laboratory, in a variety of experimental paradigms, the answer as to whether adult, embryonic stem cells, or iPS cells could serve therapeutic purposes and which source would be most efficacious, will remain unresolved. As stem cell technologies continue to evolve, it will be critical to follow how the ethical, legal, and social concerns associated with these technologies are addressed. You will find a discussion of these profoundly important issues in Module 2 and Supplement 1.

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References


  • Academy-of-Acheivement. (2010). "James A. Thomson, V.M.D. ; Ph. D." Academy of Acheivement: A Museum of Living History, from http://www.achievement.org/autodoc/page/tho1bio-1.
  • Becker, A. J., C. E. Mc, et al. (1963). "Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells." Nature 197: 452-454.
  • Choi, Y. H., A. Kurtz, et al. (2011). "Mesenchymal stem cells for cardiac cell therapy." Hum Gene Ther 22(1): 3-17.
  • Evans, M. J. and M. H. Kaufman (1981). "Establishment in culture of pluripotential cells from mouse embryos." Nature 292(5819): 154-156.
  • NIH.gov. http://grants.nih.gov/stem_cells/registry/current.htm
  • Loike, J. D. and R. L. Fischbach (2009). "Benefits of the stem cell ban." The Scientist (June 8th 2009.).
  • Shamblott, M. J., J. Axelman, et al. (1998). "Derivation of pluripotent stem cells from cultured human primordial germ cells." Proc Natl Acad Sci U S A 95(23): 13726-13731.
  • Siminovitch, L., E. A. McCulloch, et al. (1963). "The Distribution of Colony-Forming Cells among Spleen Colonies." J Cell Physiol 62: 327-336.
  • Smith, A. G. (2001). "Embryo-derived stem cells: of mice and men." Annu Rev Cell Dev Biol 17: 435-462.
  • Thomson, J. A., J. Itskovitz-Eldor, et al. (1998). "Embryonic stem cell lines derived from human blastocysts." Science 282(5391): 1145-1147.
  • Takahashi, K. and S. Yamanaka (2006). "Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors." Cell 126(4): 663-676.
  • Wang, A., K. Huang, et al. (2011). "Functional Modules Distinguish Human Induced Pluripotent Stem Cells from Embryonic Stem Cells." Stem Cells Dev. 20(11): 1937-1950


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