Where Are the Nestin Labelled Stem Cells in the Hair Follicle - Essay Example

Stem cell research is a new area of science. Before cells are used for development of new treatments, comprehensive information about their growth and behavior is obtained. Worldwide research is focused on investigating the molecular characteristics of all types of stem cells and improvement of culturing methods. Stem cells have been used in different areas of research and medicine. They are used for the replacement of damaged tissues, studying of human development, testing of new drugs, screening toxins and testing gene therapy methods. Attempts in making stem cells differentiate into specific cell types and identification of whether the specialized cells function normally have been successful with Australian scientists at the forefront of this research. Main areas of stem cell therapy research target regenerating damaged heart tissue, blood and bone marrow regeneration for improvement of bone marrow transplantation techniques and generation of safer blood cell products for patients in need of transfusion, kidney and lung disease as well as brain diseases. It has been used in understanding birth defects and in cell therapies. Immune rejection has been a major problem in transplant therapies. Efforts to overcome this problem are in progress. However, alternative strategies through use of drugs that suppress the immune system have been in use. Ethically, there have been serious objections to stem cell research since it involves the destruction of an embryo or foetus. According to many this is destruction of a potential human thus conflicting with most religious and moral views of the society. Legislation in countries such as Australia states that no embryo should be created for the purpose of stem cell research or generation stem cell lines. RESULTS The structure deer hair follicles was found to be very similar to that of the human and other mammals that have sebaceous glands attached to the upper follicle and a distinct membrane-bound dermal papilla situated at the base of the hair bulb. Anagen follicles exhibited all the normal differentiating layers to produce the outer and inner root sheaths and the hair itself. Mane hair had well developed medulla and the hair bulb around and above the dermal papilla was observed to have dividing and differentiating keratinocytes and melanocytes visibly producing pigments. Purple colored androgen receptor staining was visible in the control sections with negative controls showing no color. Skin staining was only seen in the nuclei of the dermal papilla cells of hair follicles and the outer cells of sebaceous glands. After seven days, primary dermal papilla cells began to grow out from the isolated dermal papilla explants. No evidence of epithelial contamination was observed and the dermal papilla cells displayed a different morphology to corresponding fibroblasts grown for comparison when examined under phase contrast microscopy. Morphology of cells isolated from mane or flank follicles had no difference. Dermal papilla cells had the shape of a polygon and irregular organization, they did not form aggregates. Dermal fibroblasts formed parallel arrays had an elongated shape. The rate of growth of cells from mane or flank established at the same time had no difference. Although caution is required to avoid over interpretation when using immunohistochemistry, especially across different species, this distribution replicates what has already been reported in the human hair follicle using monoclonal antibodies to the human androgen receptor. (Choudhry et al. 1992, Itami et al. 1995, DeOliveira & Randall 2000). It also concurs with the current hypothesis that androgens act on other components in the follicle via the dermal papilla (Randall 1994, 2000). DISCUSSION Hair follicles are highly dynamic organs found only in mammals. They are self-renewing structures that reconstitute themselves through the hair cycle. They have been found to maintain reservoirs of stem cells that are believed to reside in the bulge area. Keratin is a strong structural protein found in hair. Every hair strand has three layers namely; 1. The cuticle which is the outermost layer. It is thin and colorless serving as a protector for the cortex. 2. The medulla which is the innermost layer. It is only present in large thick hairs. 3. The cortex which is the middle layer. It provides strength, color and the texture for hair. Hair follicles are among the few tissues in mammals that contain stem cells with the cells interspersed within the layer at the base of the outer root sheath in a region called the bulge. Stem cells have been observed to migrate to the hair matrix where they start dividing and differentiating. The structure of hair follicles consists of the papilla, matrix, root sheath, hair fiber, the bulge and other structures. 1. Papilla It is a large structure located at the base of the hair follicle. It is made up many connective tissues and a capillary loop. Cell division in the papilla is either non-existent or rare. 2. Matrix It is located around the papilla. It consists of a collection of epithelial cells interspersed with pigment-producing cells called melanocytes. Cell division is responsible for producing cells in the hair matrix hence forming the major structures of the hair fiber and the inner root sheath. In the human body, the hair matrix epithelium is one of the fastest growing cell populations. The papilla has an ovoid shape with matrix wraps completely wrapped around it, except for short stalk-like connections to surrounding connective tissues providing access for the capillary. 3. Root Sheath It consists of an external and internal root sheath or an outer and inner root sheath respectively. The inner root sheath is consists of the Henle’s, Huxleys layer and an internal cuticle. The outer root sheath visually appears empty with the cells having a cuboid shape when stained with H&E stain. 4. Hair fiber Keratin is found in the hair fiber. 5. The Bulge It is located in the external root sheath. It has many types of stem cells supplying the entire hair follicle with new cells and healing the epidermis incase of wounds. Other structures of the hair follicle include sebaceous glands, apocrine sweat glands and the arrector pili muscles. The sebaceous glands produce sebum which is a waxy substance. Stem cells are undifferentiated cells able to differentiate into specialized cell types. They come from two main sources: 1. Embryos during the blastocyst phase of development. These cells are known as embryonic stem cells. 2. Adult tissues. These cells are known as adult stem cells. However, stem cells are of different types namely; 1. Amniotic stem cells They are found in amniotic fluid. They are very active, expanding extensively without feeders and are not tumorogenic. They are also multipotent and are able to differentiate into cells of osteogenic, adipogenic, endothelial, hepatic, myogenic, and also neuronal lines. 2. Induced pluoripotent stem cells They are reprogrammed cells given pluripotent capabilities. 3. Adult stem cells They refer to any cells derived from developed organisms. A cell must be able to divide and create another cell like itself and other cells different from itself. 4. Fetal stem cells They are derived from the organs of fetuses. The behavior of stem cells is controlled by a large number of cytokines that are produced by the cells of the dermal papilla. These cells are located in the inner and outer sheaths of the hair follicle. Androgens control hair growth by influencing the synthesis and release of cytokines from dermal papilla cells. Functions of hair in mammals include attraction of mates, retention of heat, sexual dimorphism, offering protection to the skin and absorption of sunlight. Mammalian hair follicles generate hair shafts through tightly controlled cycles of growth, remodeling, and loss. When hair follicles are made, they go through a lot of these cycles, continually making, growing and losing the hair shafts. The cycles of hair growth in all mammals comprise of three stages; 1. Anagen It is the most active stage of hair follicles. Follicle is generated and hair produced. 2. Catagen It is a short transition stage marking the end of active hair growth. Follicles undergo regression. 3. Telogen This stage can occur immaturely to humans when exposed to a lot of stress. Follicles undergo a resting phase. Visibly hairless mammals such as hippopotamuses, rhinoceroses, elephants, pigs, dolphins and whales which are all mammals are in some parts covered with very fine short hair, especially in their young ones. Genes play major roles in the formation of hair follicles; differentiation of hair shaft follows the formation of follicles. The bulge area is a region localized in the lowermost permanent portion of hair follicles. In mice and human beings, hair follicle bulge cells have been found to express nestin and present stem features as pluripotency. A class six intermediate filament protein, nestin was first described as the specific markers for CNS stem cells but recent studies suggest that it may represent a more general stem cell marker. Bulge cell characteristics had earlier been studied in mice and humans. Intermediate filament proteins are mostly expressed in nerve cells where they are implicated in the radial growth of the axon. Nestin is found in many types of cells during development. When staining is localized in the inner root sheath cells from mammals, CB1 receptor has been found to be in the proximal part of both primary and secondary hair follicles. Identification of CB1 receptor at the level of the inner root sheath may lead to the understanding of the biology of hair follicles and the possibility that cannabinoid molecules could be considered as suitable therapeutic tools in mammals. Sacpic histological staining was used in this experiment in order to enable highlighting of structures in the tissues for microscope viewing. The stains were used to define and examine bulk tissues and organelles within individual cells. Sacpic staining results were good, different parts of the hair follicle could be seen and overall results clearly showed the structure of hair follicle. However, some sections were over stained and too much blue colour was observed. Therefore, the slides were left in Celestine blue for 3 minutes instead of the recommended 5 minutes. Slides were also left in Gill’s haematoxylin for 3 minutes instead of 5 minutes. After this, the results were better and the over stain reduced. A histological method allows determination of the proportion of growing hair follicles from skin samples. Sacpic staining method modified for bulk processing produces high contrast staining of the principal tissue types present in skin. It involves adding a class-specific dye to a substrate to qualify or quantify the presence of a specific compound. Cytokeratin 5&6 is used to distinguish malignant mesothelioma from pulmonary adenocarcinoma. It has emerged as one of most useful positive markers for confirming a diagnosis of malignant mesothelioma when used in the proper way and on the appropriate setting. Anti-Goat IgG Biotin was used as the secondary antibody while performing immunohistochemistry using cytokeratins 5&6. Donkey serum was used for the whole experiment and it worked well. While performing Immunohistochemistry for the first time, the primary antibody used was Cytokeratins 5&6 and a red stain was observed on the expected areas for example outer root sheath. Concentrations 1:50, 1:40, 1:20 and 1:15 were used. Use of Concentration 1:50 showed better results compared to the other concentrations so it was concluded to be the best concentration for Cytokeratins 5&6. Immunohistochemistry is the application of specific antibodies to human tissues for diagnostic purposes. It involves the localization of antigens or proteins in tissue sections using labeled antibodies as specific reagents through antigen-antibody interactions visualized by a marker for example fluorescent dye, enzyme, or colloidal gold. All steps involved in immunohistochemistry are divided into two groups namely; sample preparation and labeling. Immunohistochemistry is used for drug development, biological research and disease diagnosis. In drug development, immunohistochemistry tests the efficacy of a drug by detecting either the activity or the down or up regulation of disease targets. Complete preparation of a sample is critical to maintain tissue architecture, cell morphology and the antigenicity of target epitopes while using right antibodies for targeting of correct antigens and amplifying of signal which is important for visualization. Tissue collected must also be quickly preserved to prevent breakdown of tissue architecture and cellular protein. Research findings show that use of immunohistochemistry may be effective in increasing patient life expectancy. Through immunohistochemistry, ability to detect antigens in tissue sections has improved mainly by countering the deleterious effects of formaldehyde with antigen retrieval and increasing sensitivity of the detection systems. Immunohistochemical staining was used to provide diagnosis of abnormal cells during the experiment; it also allowed understanding of the distribution and localization of biomarkers and the differentially expressed proteins in different parts of the tissues. Positive and negative controls are special controls run in order to test protocol and the specificity of the antibody used. A positive control tests a protocol or procedure to make sure that it works. It has always been ideal to use the tissue of known positive as a control. If a positive control tissue shows negative staining, the protocol or procedure is checked again until a good positive staining is obtained. A negative control tests the specificity of the antibody involved. No staining must be shown when omitting primary antibody or replacing specific primary antibody with normal serum. This control can be used routinely in immunohistochemical staining since it is easy to achieve. Many hair follicles change colour and size through the hair growth cycle under stimulation of androgens. In androgenetic alopecia, tiny pale hairs slowly replace large, pigmented hairs. Hair follicles can change the size and colour of the hairs they produce via their unique ability to regenerate themselves during the regular cycles of growth, regression and rest during which the hair is shed and replaced (Kligman; 1959). Observations have shown similarities in the new and previous hair. Same method of Immunohistochemistry was used for nestin experiment with the only difference being the primary antibody. Nestin was used as the primary antibody. Concentrations 1:20, 1:50, 1:75 and 1:100 were used. Concentration 1:50 and 1:75 showed better results. The best concentration was 1:75. The results showed some red stains in the connective tissues and outer root sheath. Sections were incubated with AEC and observed under a microscope, some sections took about 5 minutes to show the reaction and some about 10 minutes. It was not necessary to incubate sections in AEC for 20 minutes. However, for the nestin experiment the sections were incubated in AEC for exactly 20 minutes. Sections were stained with Gill’s haematoxylin which did not show good results since there was a lot of background stains on sections. Therefore, sections were counterstained with Harris Haematoxylin for not more than 30 seconds. According to their research (Liu et al; 2011), observed that nestin had been shown to be expressed in the hair follicle, both in the bulge area (BA) as well as the dermal papilla (DP). Previously, (Liu et al; 2011) had observed that nestin-expressing stem cells of both the bulge area and dermal papilla were pluripotent and able to form neurons and other non-follicle cell types. As reported by (Shorter et al; 2008),understanding of how hair follicles function is still limited and hair loss conditions, such as androgenetic alopecia or the putative autoimmune disease, alopecia areata ,a re currently poorly controlled . As reported by (Randall; 2007),Hormones affect follicular mesenchymal-epithelial interactions altering growing time, dermal papilla size and dermal papilla cell, keratinocyte and melanocyte activity. From research (Randall; 2008) states that These produce a waxy secretion which coats the hair under regulation by hormones,particularly androgens, and are involved in acne. According to (Shorter et al; 2008) findings indicate that human follicular dermal papillae contain K(ATP) channels that can respond to minoxidil and that tolbutamide may suppress hair growth clinically; novel drugs designed specifically for these channels could treat hair disorders. Findings by (Shorter; 2008) indicate that human follicular dermal papillae contain KATP channels that can respond to minoxidil and that tolbutamide may suppress hair growth clinically; novel drugs designed specifically for these channels could treat hair disorders. Immunohistochemical evaluation of intermediate filament nestin in other animals like dogs has led to conclusions that nestin positive cells are located in the bulge-like region of dog hair follicles and strengthening the hypothesis regarding the correlation between the bulge-like region and the dog hair follicle stem compartment. Immunostaining of skin samples collected from healthy dogs performed using a rabbit anti-nestin polyclonal antibody showed the presence of a population of immunoreactive cells in the hair follicle middle region, at the arrector pili muscle insertion level. References Landes Bioscience, 2011. The bulge area is the major hair follicles source of nestin-expressing pluripotent stem cells which can repair the spinal cord compared to the dermal papilla. [pdf] Available at: < http://www.ncbi.nlm.nih.gov/pubmed/21330787> [Accessed 18 March 2012]. The FASEB Journal, 2008. Human hair follicles contain two forms of ATP sensitive potassium channels, only one of which is sensitive to minoxidil [pdf] Available at: < www.fasebj.org/content/22/6/1725> [Accessed 18 March 2012]. Journal of Endocrinology, 2008. Stem cell factor/c-Kit signalling in normal and androgenetic alopecia hair follicles [pdf] Available at: [Accessed 16 March 2012]. Pubmed, 2008. Androgens and hair growth [pdf]Available at: < http://www.ncbi.nlm.nih.gov/pubmed/21330787> [Accessed 18 March 2012]. Mercati, F. , Pascucci, L. , Gargiulo, A. M. , DallAglio, C. and Ceccarelli, P. , 2008. Histology and histopathology, Immunohistochemical evaluation of intermediate filament nestin in dog hair follicles. [online] Available at: [Accessed 16 March 2012]. Figuera, L. E., Pandolfo, M., Dunne, P. W., Cantú, J., and Patel, P. I. 1995. Developmental biology, Mapping of the congenital generalized hypertrichosis locus to chromosome. [online] Available at: [Accessed 16 March 2012]. Hébert, J. M., Rosenquist, T., Götz, J., and Martin, G. R. 1994. Developmental biology, FGF5 as a regulator of the hair growth cycle: Evidence from targeted and spontaneous mutations. [online] Available at: [Accessed 16 March 2012]. Millar, S. E., Willert, K., Salinas, P. C., Roelink, H., Nusse, R., Sussman, D. J., and Barsh, G. S. Developmental biology, WNT signaling in the control of hair growth and structure. [online] Available at: [Accessed 16 March 2012]. Ebling, J.G., Hale, P. A.and Randall, V. A., 1992. Hormones and hair growth in Biochemistry and Physiology of the Skin. LA Goldsmith, Oxford: Claredon Press. Journal of Steroid Biochemistry, 1983. Regulation of the androgen receptor by androgen in normal and androgenresistant genital skin fibroblasts [pdf] Available at: [Accessed 16 March 2012]. Journal of investigative dermatology symposium proceedings, 1995. Interaction between dermal papilla cells and follicular epithelial cells in vitro: effect of androgen [pdf] Available at: [Accessed 16 March 2012]. Read More