Definition

Cellular differentiation is the normal process by which a cell becomes increasingly specialized in form and function. The classic example is the process by which a zygote develops from a single cell into a multicellular embryo that further develops into a more complex fetus. Cellular differentiation is regulated by many processes and substances including cell size, shape, polarity, density, metabolism, and extracellular matrix composition. Under the influence of these external factors, each cell is programmed to differentiate and eventually mature into its specialized cell such as heart, muscle, skin, and brain cells.
Helen Blau Helen M. Blau is the Donald E. and Delia B. Baxter Professor, the Director of the Baxter Laboratory in Genetic Pharmacology, and the Director of Gene Therapy Technology at Stanford University School of Medicine. Her research centers on how cells maintain quiescent, proliferative, or their differentiated states. Perturbing the intracellular milieu allows the Blau lab to probe regulatory networks that determine cell fate. This knowledge directly sheds light on issues of stem cell quiescence, self-renewal, differentiation, and carcinogenesis and in turn has important implications in the field of regenerative medicine.

 
















There have been dramatic changes in our understanding of cellular differentiation. Over 20 years ago, Helen Blau and colleagues generated stable heterokaryons by fusing terminally differentiated human fibroblasts with terminally differentiated mouse muscle cells. Using this technology, Dr. Blau and colleagues, found that the heterokaryons they had created retained some plasticity as evidence by their capacity to synthesize human muscle proteins (Pomerantz, Mukherjee et al. 2009).

Thought Question


How do scientists classify a unique specialized cell type?


Dolly


Other investigators have shown that In the presence of appropriate biological substances, differentiated cells from one lineage can be triggered to express genes from another lineage. These experiments, prior to 1997, suggested that in mammalian cells, differentiation can be reprogrammed.

In 1997, one of the most exciting insights into differentiation emerged from the cloning of Dolly the sheep (Wilmut, Schnieke et al. 1997). The cloning of Dolly from terminally differentiated adult mammary cells shattered the existing dogma that cell differentiation was irreversible.


Dolly (1996-2003)


Dolly the sheep was the first mammal cloned from an adult somatic cell, specifically a mammary cell, using nuclear transfer technology. Ian Wilmut, Keith Campbell, and others at the Roslin Institute were leaders of this groundbreaking project.


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The cloning process that produced Dolly.






















What does pluripotency really mean?

Pluripotency refers to a cell’s capacity to differentiate into any of the three germ layers: endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenitals), or ectoderm (epidermal tissues and nervous system). In contrast to totipotent cells, pluripotent cells cannot develop into a fetus or adult animal because they lack the potential to contribute to extra-embryonic tissue that forms the placenta. Though this definition sounds simple, it is actually quite difficult to determine experimentally whether a particular human stem cell line has the requisite developmental capacity to meet all the criteria of pluripotency.

And to complicate matters, subpopulations of human embryonic stem cells (ESC) have quite distinct capacities to differentiate in vitro or in vivo. Thus, the tissue source of pluripotent stem cells is another critical factor that scientists must consider in differentiation. A stem cell derived from an embryo may require different signals than a stem cell derived from an adult somatic cell to program it to differentiate into a specialized cell type. Another complication is related to the fact that some stem cells can spontaneously transform into cancer cells while others cannot.

Understanding these complicated issues is critical for scientists as they develop new stem-cell based therapeutics for diseases such as cancer, spinal cord injury, Parkinson’s disease, Alzheimer’s disease, and diabetes (Pera 2010).
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Embryonic stem cell differentiation

Embryonic stem cells have been the cell model for our understanding of the mechanisms regulating cell differentiation. As discussed in Module 1, stem cells have two basic properties:
  1. The capacity for self renewal of their stem cell state and
  2. The ability to differentiate into a wide variety of specialized cell types.

Maintaining the balance specifically between stability and plasticity has been a major challenge for stem cell scientists. Over the past several years, extensive studies have been conducted that focus on understanding the contributions of transcription factors and epigenetic enzymes to the "stemness" properties of these cells. Identifying the molecular switches that regulate ES cell self-renewal versus differentiation could provide insight into the nature of the pluripotent state while also enhancing the potential use of these cells in therapeutic applications.

How do epigenetic processes, such as transcription factors and DNA modification regulate the activity of genes in the cell?


There are several ways in which the cell can regulate which genes are turned on (to synthesize a specific protein that is involved in the cellular activities) and which genes are turned off (silenced). One pathway involves transcription factors and the other pathway involves chemical modifications of either DNA or the proteins associated with DNA, i.e., histones (Hanna, Saha et al. 2010). The processes we mention are part of epigenetics which we describe below.

Clearly, the properties of different specialized cells depends on which genes are turned off or on in the cells. Thus, a muscle cell will have a different array of genes turned on or turned off that will dictate its capacity to function as a muscle cell. A white blood cell, in contrast, will have a different array of genes than in a muscle cell to enable it to serve as an immune cell.
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Transcription factors, DNA modifications, and epigenetics

Transcription factors

Transcription factors are critical components that control the "stemness" properties of these cells. Three transcription factors, OCT4, SOX2, and NANOG, cooperate to ensure the self-renewal and pluripotency of ES cells (Boyer, Lee et al. 2005; Loh, Wu et al. 2006). These factors are highly expressed in undifferentiated ES cells. In addition, chromatin methylation is carried out through two major repressive pathways: Polycomb group (PcG) repressive complexes and promoter DNA methylation, both of which can modulate the fate of ES cells (Christophersen-and-Kristian-Helin 2010).

DNA modifications

Chemical modification of DNA and histones (proteins that surround DNA) occurs frequently during stem cell differentiation in regulating gene activity. Methylation of specific sites in the sequence of DNA is a common mechanism for a cell to turn off protein synthesis of a specific protein. In human cells, chemical modifications, such as DNA methylation, typically occur at the cytosine base pair of DNA. In addition, chemical modification of the proteins that surround DNA, such as histones, is another method for cells to regulate the activity of specific genes. Methylation of the N-terminal tails of histone proteins that surround the DNA molecules has been shown to be an effective method to silence a gene.

Epigenetics

Epigenetics is a hereritable process and differs from Mendelian genetics. In Medelian genetics changes in the base pair sequence of a gene can be a critical determinant of its activity. At first glance epigenetic trans-generational inheritance of acquired characteristics is reminiscent of a theory of genetics proposed by Jean-Baptiste Lamarck [for example, the giraffe acquired a long neck over generations because of evolutionary pressure to reach the highest branches to obtain its natural food, leaves; i.e., they stretched their necks all the time and gradually this led to changes in the tissues (muscle, bone) that resulted in longer necks.) In contrast, Darwinian theory of natural selection would instead say that shorter-necked giraffes, unable to reach leaves in specific habitats, would die out, and long-necked giraffes would become the dominant type because they survived and passed on their genes. In fact, the current underlying mechanisms of epigenetics provide scientific evidence how environment can trigger heritable changes. There is ample evidence in animals and even in human beings that environmental factors shape health and disease via epigenetic mechanisms that mediate gene-environment interactions. According to Dr. Moshe Szyf, a leading geneticist, epigenetics is a physiological mechanism by which the genome senses the world and changes itself (Berreby 2011).

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Epigenetic mechanisms (Source: Wikimedia Commons)


Dr. John Greally on epigenetics


For more information on epigenetics, view the following videos produced by Albert Einstein College of Medicine/Montefiore Medical Center, featuring Dr. John Greally.

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Transcription factors in cellular differentiation


Further in-depth analysis of DNA methylation - polycomb group in ES cells

Genome-wide approaches revealed that the transcriptionally silent developmental genes targeted by OCT4, SOX2, and NANOG in ES cells are also occupied by Polycomb group repressive complexes (Boyer, Plath et al. 2006; Lee, Jenner et al. 2006). Polycomb group proteins facilitate maintenance of cell states through gene silencing. They were first identified in the Drosophila fruit fly as a result of their essential roles as repressors of body patterning genes, including homeobox (HOX) genes during fruit fly development (Lewis 1978; Struhl 1981). HOX genes are expressed in distinct domains along the body axis and act to give cells of diverse tissues their unified regional cell identities.
The Polycomb group proteins are required for early mammalian embryo development but not for maintaining ES cell pluripotency.

Polycomb group mutant ES cells can still
  • self-renew
  • maintain normal morphology
  • express OCT4, SOX2, and NANOG
  • but do not differentiate efficiently into the three germ layers.

A key question of importance for ES cell self-renewal and differentiation concerns how Polycomb group proteins binding and dissociation to chromatin can be regulated. This question is critical because the Polycomb group proteins themselves do not have the ability to bind DNA-specific sites. Thus, recruitment of Polycomb group proteins is believed to require an interaction with sequence-specific transcription factors. The availability of these transcription factors or competing transcription factors may be involved in regulating the sustained binding of Polycomb group proteins to their target genes.


Conclusions


  • Chemical modifications of DNA and DNA - associated proteins play a critical role in regulating cellular differentiation.
  • Molecular maps of chemical modifications of DNA and its proteins will eventually be useful in identifying the differentiating state of a cell.
  • One of the most significant outcomes in cloning technology has revealed that cellular differentiation is a reversible process. The creation of the sheep Dolly illustrated how this is accomplished.


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References


  • Berreby (2011). “Environmental Impact".
  • Boyer, L. A., T. I. Lee, et al. (2005). "Core transcriptional regulatory circuitry in human embryonic stem cells." Cell 122(6): 947-956.
  • Boyer, L. A., K. Plath, et al. (2006). "Polycomb complexes repress developmental regulators in murine embryonic stem cells." Nature 441(7091): 349-353.
  • Christophersen-and-Kristian-Helin (2010). Epigenetic control of embryonic stem cells fate, The Rockefeller University Press, doi: 10.1084/jem.20101438 © 2010.
  • Dolinoy, D. C. (2008). "The agouti mouse model: an epigenetic biosensor for nutritional and environmental alterations on the fetal epigenome." Nutrition Reviews 66 Suppl 1: S7-11.
  • Hanna, J. H., K. Saha, et al. (2010). "Pluripotency and cellular reprogramming: facts, hypotheses, unresolved issues." Cell 143(4): 508-525.
  • Hollar, S. (2011). A Closer Look at Biology, Microbiology, and the Cell, Rosen Education Service.
  • Hollingsworth, J. W., S. Maruoka, et al. (2008). "In utero supplementation with methyl donors enhances allergic airway disease in mice." J Clin Invest 118(10): 3462-3469.
  • Lee, T. I., R. G. Jenner, et al. (2006). "Control of developmental regulators by Polycomb in human embryonic stem cells." Cell 125(2): 301-313.
  • Lewis, E. B. (1978). "A gene complex controlling segmentation in Drosophila." Nature 276(5688): 565-570.
  • Loh, Y. H., Q. Wu, et al. (2006). "The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells." Nat Genet 38(4): 431-440.
  • Pera, M. F. (2010). "Defining pluripotency." Nat Methods 7(11): 885-887.
  • Pomerantz, J. H., S. Mukherjee, et al. (2009). "Reprogramming to a muscle fate by fusion recapitulates differentiation." J Cell Sci 122(Pt 7): 1045-1053.
  • Struhl, G. (1981). "A gene product required for correct initiation of segmental determination in Drosophila." Nature 293(5827): 36-41.
  • Wilmut, I., A. E. Schnieke, et al. (1997). "Viable offspring derived from fetal and adult mammalian cells." Nature 385(6619): 810-813.



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