Stem Cells (10 slides)

Stem cells occupy a special place in the development of animal systems, and are of especial interest in regard to human physiology. This can be understood from the observation that the entire organism and its individual parts develop from only a very few stem cells that, in turn, are formed by the fertilized ovum. In developing embryos, cells acquire their various specializations through the process of cell differentiation, developing subsequently into the various specific tissues and organs of the body. The seven day-old human pre-embryo, for example, is composed of several thousands individual cells that will each take on a specific role in the developing human body. The specific functions of these cells are determined by their relative location in the cell mass, and it has been observed that, in the pre-embryo stage, cells that are relocated from their original positions will adopt the function corresponding to their new location. This flexibility is lost as the embryo develops.
Since their initial isolation by James A. Thomson at University of Wisconsin, stem cells were believed to be a feature only of the pre-embryonic stage and equivalent to the human embryo. That is to say, each embryonic stem cell was in essence a potential human being. This view posed a basic moral dilemma for stem cell researchers and policy-makers alike. Since 1996, the United States federal budgets, through which a great deal of medical research is funded, have prohibited “the use of taxpayer money for “research in which a human embryo or embryos are destroyed, discarded or knowingly subjected to risk of injury or death.”” (Johnston 2010). Criticism of this statement, known as the Dickey-Wicker Amendment, is aggravated by the manner in which researchers and investors work around the moral question by relying on a very narrow legal interpretation that takes advantage of the imprecise wording, and comparison to regulations governing stem cell research in other countries that have stated very clearly what is and is not permissible under the corresponding regulations.
The very characteristic that has made stem cells so 'taboo' for research purposes is, paradoxically, the very characteristic that makes their use and research based on them so very promising. Stem cells come in two basic types: unipotent and pluripotent. Stem cells divide asymmetrically to produce a daughter stem cell and a sibling daughter cell that is slightly more specialized and has lost some of its developmental potential. Unipotent stem cells produce only one type of differentiated daughter cell, while pluripotent stem cells may produce two or more types of differentiated daughter cells. It is the pluripotent type that has the greatest promise for clinical use, especially if they are stem cells that have been extracted from embryos, or “embryonic stem cells”, typically abbreviated in the literature as ESCs. ESCs have the remarkable ability to differentiate into any cell type depending on the chemical instructions they receive in their growing environment. In principle, this means that they could be used for transplantation treatments, in which they would act as replacements for damaged or defective tissues. The caveat to this sort of application, however, is that the undifferentiated transplanted cells must be given exactly the correct differentiation instruction, else they present a serious risk for the formation of tumors and teratomas. Some recent research has addressed this issue with the discovery of an antibody that binds specifically to undifferentiated ESCs resulting in their destruction through the mechanism of oncosis (Choo et al, 2009). In oncosis, the cell membrane of undifferentiated stem cells breaks down through the formation of a large number of pores. The ability to apply this principle selectively to stem cell transplants has the potential to eliminate the likelihood of tumer and teratoma formation.
The innate versatility of ESCs has been found to be an active state requiring the active, coordinated effort of several genes that encode cellular maintenance factors necessary for the stem cells to persist as stem cells rather than to mature. The effect is reversible, and the same genes can be used to “reprogram” adult cells, inducing them to revert to a pluripotent state in which they are essentially indistinguishable from ESCs (Chan et al, 2009). Such cells are referred to as “induced pluripotent stem cells”, or iPSCs. Recent research has demonstrated that somatic cells from various tissues, for example skin cells, can be harvested by very simple means and subsequently reprogrammed to express genes that will reverse the differentiation process, producing iPSCs. Theoretically, these cells can then be instructed to re-differentiate into any mature cell type. The major benefit of this approach is that it would enable derivation of tissues specific to an individual, and perhaps even the production of whole organs for transplant purposes (Feng et al, 2012). Such organs would presumably be entirely free of the possibility of rejection, being physiologically indistinguishable from the parent organ being replaced.
Human ESCs (hESCs) also have the property of being able to undergo numerous cycles of cell division without changing their undifferentiated state. This capability for self-renewal facilitates the growth and multiplication of hESCs from an initial colony of relatively few cells to a much larger quantity. Using an RNA interference screening method, it has been found that this property is governed by some 566 different genes (Chia et al, 2010). Research has demonstrated that the molecular basis for undifferentiated self-renewal depends on the phosphorylation of tyrosine residues in hESCs. Fibroblast growth factor 2 (FGF-2) is known to activate tyrosine kinases that are principal components of the biochemistry that control hESC self-renewal and differentiation (Ding et al, 2010; 2011).
The processes that drive the differentiation of hESCs into various mammalian cell types is a complex mystery , though recent studies have demonstrated that as hESCs are exposed to different proteins at different times, they are signalled to transform or differentiate from simple cells into specialized cells. The early stage of differentiation is apparently triggered by the signal presented by the presence of the two proteins Nodal and Activin. The strength of the signal, which in turn depends on the amounts of the two proteins present in the growth environment, plays a very significant role in the differentiation mechanism. The proteins are detected by receptors on the surface of the cell membrane, triggering a response within the cell that affects the functioning of the DNA within the cell. The mediator of the response within the cell is the protein known as Smad2 (Lee et al, 2011; Teo et al, 2011)
It is important to know what effect there might be from maintaining hESCs through several cycles of self-renewal, and especially what is the potential for the introduction or occurrence of mutations in the genome of the hESCs that might result in the formation of cancerous growths rather stable, normal tissue. That this potential exists has been identified in certain hESC lineages (ISCI, 2011), and underscores the degree of complexity and level of risk that characterize the entire biological system of stem cells and stem cell research in general.
Given that ESCs have enormous medical potential, another technical aspect of that potential is in how to exploit ESCs effectively. This requires determining methods of effectively increasing the number of ESCs and the most effective scaffolds and matrices on which to grow the desired tissues. These are the areas of biomaterials and bioengineering. Research has been carried out to identify materials and methods for encouraging PSC proliferation, using a three-dimensional fiber scaffold of polymeric material produced by the interaction of chitin and sodium alginate (Lu et al, 2012). The material proved effective at both proliferating and storing PSCs without any genetic mutations. Another approach uses the target organ's own structural framework of collagen proteins and growth factors that remain after the other tissue material have been removed (Ng et al, 2011). This method has potential value, but there are many problems to be overcome. A recent study (Kumar et al, 2011) demonstrates that there is tremendous promise in the development of suitable materials and media for the growth of human induced pluripotent stem cells (hiPSCs) to produce specific tissues. Stem cells are found in essentially all tissues, and this particular study demonstrated the intimate role played by distal airway stem cells (DASCs) in the repair and regeneration of lung tissue that had been damaged by Acute Respiratory Distress Syndrome triggered by the H1N1 influenza virus. The growth of hiDASCs in a three-dimensional growth medium produced alveoli-like structures, suggesting that hiPSC therapies might be used to treat conditions in which the airway has been damaged. That hiPSCs are capable of therapeutic application has been readily demonstrated by a device termed the “skin gun”, the use of which in the treatment of severe burns has been documented. The device is loaded with a solution of stem cells which is then sprayed over the area damaged by the burn. Remarkably, in merely two weeks all visible sign of skin loss or damage from the burn is eliminated and the area is encased in an essentially blemish-free and fully functional epidermal layer.
The ability to culture hiPSCs in an undifferentiated state and then allow them to differentiate into specific cell types has great potential for developmental and tissue differentiation studies, for disease modelling, drug development and, perhaps greatest of all, regenerative medicine. Undifferentiated hiPSCs can be maintained and proliferated on either feeder-dependent or feeder-free culture systems, the former using fibroblast cells as the feeders. The use of feeder cells can be labour intensive, however, and carries the risk of transmitting certain pathogens to the hiPSCs, which would severely interfere with their clinical application. HiPSC cultures have a natural tendency to differentiate, so maintaining undifferentiated hiPSC cultures demands specialized techniques in order to achieve a balance between promoting pluripotency and inhibiting spontaneous differentiation of the stem cells.

References:
Chan, K. K-K., Zhang, J., Chia, N-Y., Chan, Y-S., Sim, h.S., Tan, K.S., Oh, S.K-W., Ng, H-H. and Choo, A.B. “KLF4 and PBX1 directly regulate NANOG expression in human embryonic stem cells” Stem Cells 27: 2114 – 2125 (2009)
Chia, N.Y. et al “A genome-wide RNAi screen reveals determinants of human stem cell identity” Nature 468: 316 – 320 (2010)
Choo, A.B., Tan, H.L., Ang, S.N., Fong, W.J., Chin, A., Lo, J., Zheng, L,Hentze, H., Philp, R.J., Oh, S.K. et al “Selection against undifferentiated human embryonic stem cells by a cytotoxic antibody recognizing podocalyxin-like protein-1” Stem Cells 25: 1454 – 1463 (2008)
Ding, V.M.Y. et al “Tyrosine phosphorylation profiling in FGF-2 stimulated human embryonic stem cells” PloS ONE 6: e17538 (2011)
Feng, B., Jiang, J., Kraus, P., Ng, J-H., Hrng, J-CD., Chan, Y-S., Yaw, L-P., Zhang, W., Loh, Y- H, Han, J. et al. “Reprogramming of fibroblasts into induced pluripotent stem cells with orphan nuclear receptor Esrrb” Nature Cell Biology 11: 197 – 203 (2009)
International Stem Cell Initiative “Screening a large, ethnically diverse population of human embryonic stem cells identifies a chromosome 20 minimal amplicon that confers a growth advantage” Nature Biotechnology 29: 1132 – 1144 (2011)
Johnston, Josephine “America's Stem Cell Mess” The Scientist Vol. 24, No. 10 (October 2010):33 – 34
Kumar, P.A. et al “Distal airway stem cells yield alveoli in vitro and during lung regeneration following H1N1 influenze infection” Cell, 147: 525 – 538 (2011)
Lodish, Harvey, David Baltimore, Arnold Berk, S. Lawrence Zipursky, Paul Matsudaira and James Darnell (1995) Molecular Cell Biology New York, NY: W.H. Freeman and Company
Lu, H.F/. Narayanan, K., Lim, S-X., Gao, S., Leong, M.F. And Wan, A.C.A. “A 3D microfibrous scaffold for long-term human pluripotent stem cell self-renewal under chemically defined conditions” Biomaterials 33: 2419 – 2430 (2012)
Ng, S.L., Narayanan, K., Gao, S. and Wan, A.C. “Lineage restricted progenitors for the repopulation of decellularized heart” Biomaterials 32: 7571 – 7580 (2011)
Tan, H.L., Fong, W.J., Lee, E.H., Yap, M. and Choo, A. “mAb 84, a cytotoxic antibody that kills undifferentiated human embryonic stem cells via oncosis” Stem Cells 27: 1792 – 1801 (2009)
Teo, A.K.K. et al “Pluripotency factors regulate definitive endoderm specification through eomesodermin” Genes and Development 25: 238 – 250 (2011)
The Skin Gun http://channel.nationalgeographic.com/channel/explorer/videos/the-skin-gun/

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