Lab 2 attended though didn't know we had to do attendance on our page
Lab Task 1; Research Paper Summaries
Li M, Zhao H-C, Li R, Yu Y, Qiao J (2014) Chromosomal Aberrations in In-Vitro Matured Oocytes Influence Implantation and Ongoing Pregnancy Rates in a Mouse Model Undergoing Intracytoplasmic Sperm Injection. PLoS ONE 9(7): e103347. doi:10.1371/journal.pone.0103347
In-vitro fertilisation (IVF) and In-vitro maturation (IVM) are potential ways to alleviate issues with gonadotrophin stimulation. IVF has been accepted as the prefered Assisted Reproductive technique because of the higher success rate then IVM, which has 7 to 12% probablitly of plantation. Previous studies have concluded that the low success rate is related to the etiology of infertility rather then the technique itself. The selection of specific oocytes based on morphological features can greatly increase chance of implantation.
The study goal is to analyse spindle and chromosome and spindle configurations during the maturation process and resulting pre- and post-implantation embryos.
Experiment 1: used immature oocytes collected and matured in-vitro, with 3 cultures one at 18, 20 and 22 hours. alpha-tubulin and chromosome configurations were imaged by immunofluorescnce method. Experiment 2: used intracytoplasmic sperm injection to fertilise IVM oocytes, the pre-implanation phase was then compared to that of fertilised IVo oocytes. Expierement 3: Resulting embryos where introduced to a Pseudo-pregnant mouse, followed by dissection of fetuses on days 6.5 and 12.5.
Results; Spindle assembly in expiement 1 showed 18h had less that 50% originally, then rose to 52% in metaphase,the 20 and 22 hour grounds have significantly higher abnormalities (p<0.05) though had lower then a fresh control group. In expierment 2 1072 immature oocytes were collected and cultured with 924 becoming mature at MII stage, with 18h and 20h groups having reduced gene expression compared to 22h and control. Experiment 3 no significant difference in embryos on day 6.5, with 18h having slightly lower fetal rate then other groups. Day 12.5 showed a significantly reduced implantation rate in 18h group in comparison to control and 22 h groups. Full term development results average number of pups 18h=3, 20h=4.5, 22h =4.4, IVO=5.88
Shows greater chance of chromosome abnormalities in embryos from IVM timing and the relationship with post-implantation development in mouse models.
Shi W, Xu B, Wu L-M, Jin R-T, Luan H-B, et al. (2014) Oocytes with a Dark Zona Pellucida Demonstrate Lower Fertilization, Implantation and Clinical Pregnancy Rates in IVF/ICSI Cycles. PLoS ONE 9(2): e89409. doi:10.1371/journal.pone.0089409
Previous studies have shown abnormal morphology of oocytes and embryos results in lower viability for pre-implantation embryos and increase early pregnancy loss. This study focused on one specific oocyte abnormality a Dark Zona Pellucida (DZP)which previous studies have concluded to not significantly impact fertilization, embryo quality and pregnancy rate. Unlike previous studies DZP embryos would be seperated from NZP (Normal Zona Pellucida)
Patient population consisted of 268 infertile couples aged less than 38 years, being treated with IVRF or ICSI64 patients were put on embryo transfer cycles with occyte surronded by DZP being further subdivide based on percentage of Dark Zona pellucida; Group A(47) had 58% of Zona Pellucida dark on average and Group B(22) had entierly Dark Zona Pellucida. The remaining 204 patients made up the control group with NZP.
All patients were stimulated using a long pituitary down -regulation protocol. Oocytes were cultured in fertilisation medium. Sperm was used according to density gradient. Fertilized oocytes were placed in cleavage medium. Twenty mature oocytes (10 NZP, 10 DZP) were examined using JEOL-1230 Transmission Electron Microscope.
Reuslts; Miscarriages and live births for the three groups had no large differences. Though there were marked differences in fertilisation rate, frequency of high quality embryos, implantation rates and clinical pregnancy rates. All being lower Group B compared to control. Comparing the microscopic morphology shows no change in thickness of Zona Pellucida. NZP showed typical typical mitochondria shape; oval to spherical in shape with dense matrix and few cristae. DZP Mitochondria displayed swollen mitochondria encircling or containing vacuoles.
Study concluded that higher DZp results in decreased fertilisation rate, low rate of high quality embryos, adverse pregnancy outcomes and increased abnormal mitochondria and cytoplasmic vacuoles.
Lab Task 2; Image upload
Fetal Skeletal Muscle Progenitors []
Sakai H, Sato T, Sakurai H, Yamamoto T, Hanaoka K, et al. (2013) Fetal Skeletal Muscle Progenitors Have Regenerative Capacity after Intramuscular Engraftment in Dystrophin Deficient Mice. PLoS ONE 8(5): e63016. doi:10.1371/journal.pone.0063016
Lab Task 3
Julie R. Fuchs, Shinichi Terada, Didier Hannouche, Erin R. Ochoa, Joseph P. Vacanti, Dario O. Fauza.Engineered fetal cartilage: Structural and functional analysis in vitro. Journal of Pediatric Surgery Volume 37, Issue 12, Pages 1720–1725, December 2002
Sayer AA1, Cooper C.Fetal programming of body composition and musculoskeletal development.Early Hum Dev. 2005 Sep;81(9):735-44.
Baróti B1, Pap Z, Pánti Z, Buruian MM, Pávai Z. Morphometric and ultrasonographic study of the human fetal hip joint during intrauterine development. Rom J Morphol Embryol. 2013;54(4):977-81.
Liberty G1, Boldes R, Shen O, Shaul C, Cohen SM, Yagel S. The fetal larynx and pharynx: structure and development on two- and three-dimensional ultrasound. Ultrasound Obstet Gynecol. 2013 Aug;42(2):140-8. doi: 10.1002/uog.12358. Epub 2013 Jul 16.
Lab Task 4
Identify a paper that uses cord stem cells therapeutically and write a brief (2-3 paragraph) description of the paper's findings.
Stem cell therapy and curcumin synergistically enhance recovery from spinal cord injury. []
This paper investigates the result of Stem cell micro injection on spinal cord injury recovery and the effect curcumin(anti-inflammatory form turmeric) on Stem Cell Proliferation.
Findings; The addition of low concentrations of curcumin(500nM) results in a 180% increase in neurosphere proliferation,concentrations equal or higher then 1um fragements neurospheres and causes apotptosis. BBB Scores for Moderate SCI; Scores in week 5 and 6 showed a 33% higher score in treated rats compared to control. There was no significant difference between NSC and NSC/curcumin groups. BBB Scores for Severe SCI; After 2 weeks NSC and NSC/curcumin show similarly significant improvement compared to control, this improvement degrades in later weeks. Body weight all rats showed a decrease in weight then increase corresponding to recovery, except in Severe SCI in which curcumin alone showed improvement in week 5. Soleus Muscle Mass; in Moderate SCI showed no significant differentiation, in Severe SCI all treatemnt groups had higher mass then control particularly combination NSC/curcumin. Histopathological analysis;NSC and NSC/Curcumin displayed better recovery by comparing spared area to total area
Study displays Neural stem Cell therapy with curcumin is effective in recovery in Spinal cord injury. In the case of Moderate SCI effect of NSC and NSC/Curcumin showed a similarly significant recovery increase. In Severe SCI NSC/Curcumin had a greater aid in recovery. Curcumin assists recovery by reducing inflammation and gliosis, allowing unobstructed increased availability of neurotrophic factors from neural stem cells. The increased proliferation in-vitro supports the notion proliferative properties of curcumin.
There are a number of developmental vascular "shunts" present in the embryo, that are closed postnatally. Identify these shunts and their anatomical location.
The Cardiovascular system has three important shunts present in the developing embryo, allowing blood to flow to differ from typical postnatal circulation. The key developmental shunts are Foramen Ovale,Ductus Venosus, Ductus Arteriosus.
Foramen Ovale; present in the Interatrial septum connecting the Left atrium and Right atrium. Allows oxygenated blood to bypass the lungs and enter the arterial network immediately. Usually open at birth, soon closed by two flaps of interatrial setum forming the fossa ovalis.
Ductus Venosus; connection of the umbilical vein and portal vein and inferior vena cava, purpose is to allow blood to bypass the liver. Functionally closes at birth and structurally closes within first week.
Ductus Arteriosus; connection of pulmonary artery and proximal ascending aorta, become ligamentum arteriosum after closure at birth
--Mark Hill Review of paper is not terribly clear you should spend more time with this. Three shunts are correct (3/5)
Lab Task 5
Anorectal Malformation is a defect of the anus and rectum which occurs during fetal development. There are multiple classifications of anorectal malformation based on the associated fistula. Low lesion;colon lies close to skin with an resulting in narrowing of the anus and rectum ending in blind pouch. High lesion; anus opens into bladder or genitalia, ,Persistent cloaca; rectum, vagina and urethra are joined into a single channel. 
Causes; Abnormal development during weeks 7-10 as the lower section of intestinal tract forms, common associated with abnormal Sonic Hedgehog signalling. Originally the lower section large intestine and urinary tract are a single mass of cells. during the 7th-10th week this single mass of cells differentiates, seperate and form their appropriate organs. There is no correlation between activity of mother during pregnancy and incidence of malformation, more common in boys then girls.
Anorectal malformation occurs in 1 of 5,000 babies. Is associated with multiple other conditions and disorders these include; spinal , congenital, tracheal, esophageal, kidney, urinary and limb defects 
--Mark Hill These are good papers for the abnormality of anorectal malformation. (5/5)
Lab Task 6
Identify and write a brief description of the findings of a recent research paper on development of one of the endocrine organs covered in today's practical.
Hick, A., Delmarcelle, A., Bouquet, M., Klotz, S., Copetti, T., Forez, C., Van Der Smissen, P., Sonveaux, P., Collet, J., Feron, O. and others, (2013). Reciprocal epithelial: endothelial paracrine interactions during thyroid development govern follicular organization and C-cells differentiation. Developmental biology, 381(1), pp.227--240. 
Reciprocal epithelial: endothelial paracrine interactions during thyroid development govern follicular organization and C-cells differentiation
Study investigates how endothelial cells are recruited into the developing thyroid, and wether they control thyrocytes, C-cell differentiation and glandular organization. Thyroid explants were micro dissected from Vegfa-floxed and Pax8-cre mice embryos, then cultured in gelatin-coated dishes. Embryos were fixed and RNA probed for Vegfa and VEGF. Dissected thyroids were fixed in formaldehyde and some sections were immunofluorescence. Total RNA was extracted from embryonic thyroid lobes and underwent Real time polymerase chain reaction. Control and cKO lobes were rinsed and stained with uranyl acetate and examined under an electron microscope.
Results; Through out developmental stages of Endothelial cells closely surround thyroid epithelium; VEGFA knockout mice die around the period of childbirth, Reduction in vascular density hinders reorganization of epithelial mass into mono-layers because of polarization(shown by lack of basal pole). Additionally the thyroid developmental processes of bilobation, differentiation, polarization and vascularization are reproducible in a invivo culture, if adequate oxygen and nutrients are supplied
Findings; VEGFA is increased in a hypoxic environment via Hypoxia-induced factor-1 (HIF1A). When polarity normalizes decreases VEGFA expression. VEGFA derived from epithelium recruits the dense endothelial network in thyroid and promotes expansion. Endothelial cells are crucial for reorganization of follicular cells into follicles and differentiation of C-cells.
Continual interaction of epithelial and endothelial cells determines extent of thyroid development.
Identify the embryonic layers and tissues that contribute to the developing teeth.
Embryonic layers; Mesoderm, Ectoderm, neural crest ectomesenchyme all contribute to teeth development;
Ectoderm; contributes to Ameloblasts which later form tooth enamel
Neural crest mesenchyme; contributes to odontoblasts which undergoes dentinogenesis forming dentin
Mesoderm: forms blood supply of dental pulp
--Mark Hill Fine (5/5)
Lab Task 8
Indifferent development of gonads Originates from intermediate mesoderm forming genital ridges, pronephros appears with pronephric duct and nephrogenic mesenchyme, mesonephros appears with mesonephric tubule. Both later will fully or partially degenerate. Gonads descend down to pelvic region.
Gonadal differentiation Presence of Y chromsome has 48 protein-coding genes which begin the differentiation of the gonads into testes. In around week 6 Sertoli cells begin the appear in the primordia, to instruct germ cells; which have migrated through the primative streak. Leydig appear begin to secrete testosterone. The germ cells are enveloped in Sertoli cells which stop thier differentiation at T1 prospermatogonia until after birth.
In the testes the paramesoneph degenerates because of anti0Mullerian hormone secreted by the Sertoli cells. Mesonephric duct remains and under testosterone further differentiates to later form rete testis and ductus deferens. Testis cords of Sertoli and Germ Cells differentiate into semiferous tubules.
Semififerous tubule  External genitalia differentiation Indifferentiation stage in which cloaca is divided by proliferating mesencyme forming the urorectal septum. The presence of Dihydrotestosterone result in growth of genital tubercle and formation of genital folds. The folds are maintained then fuse creating perineal and penile raphe. Lateral to urethral folds is the labioscrotal swelling which later becomes the scrotum
--Mark Hill 5
Lab Task 9 - Peer Reveiws
Group 1 Respiratory
The pages structure is quite well done, with appropriate use of heading. The introduction explains the general development of the respiratory system, differentiating embryonic, fetal and postnatal time-span. Like division of conducting and respiratory zones with strong general description of each zones components. The referencing of the first three heads content and images was not present though I presume this could be easily resolved. The Development stages table gives is very informative, particularly liked how the stages during the fetal period had more in-depth feature description then the embryonic and postnatal stages.
The Current research and findings section was in-depth with strong explanations and in-text referencing; only part lacking referencing is the Functional unit section. Some sentences should be broken up to avoid excessive use of commas “However, a study conducted…”. There seems to be a picture missing or placed incorrectly for one of the 2013 studies. The Lung Model picture is relevant but with no caption, though the description and referencing when clicking on the image is solid. Little improvement is needed for the Historical findings and Abnormalities sections, great referencing and content. Particularly impressed with the sheer amount of abnormalities presented, with information being sourced from 2-3 references for each abnormality.
To improve referencing needs to be added particularly to the introduction, conduction and respiratory zone. The references need to be collected at the bottom of the page instead of after every couple of sections. The removal of the multiple reference subheadings would make the content and page in general easier to navigate. Many of the earlier images should be captioned properly and referenced properly, with missing info like ((Template: Student Image)), description, copyright info. The content of Respiratory and Lung Development Stages could be slightly more in-depth though not too significant of an issue. Overall content is written well, providing information on all the important objectives, only place improvement is properly required is referencing and some formatting.
Group 2 Respiratory
Nice introduction addressing function of the system to set the tone, more detail in the development of components would be preferable. Abnormalities part of introduction has sufficient detail and referencing. The description of anatomical and histological structure described is mostly post-natal, more linking the structure to embryonic beginning and fetal continuation of development. Historic findings heading should be removed or expanded on, searching for articles before a certain period could be helpful. Introduction is easy to understand and presents the structure of the page exceptionally.
Developmental timeline is good at giving the reader a simple overview of renal development, putting it into a table would make it look more professional. The Current research model section gives readers much insight into the method developmental stages were discovered. I would recommend find additional 1-2 methods.
The Development section is divided into four structural components, greatly increasing ease of understanding. The information presented in this section is highly informative, well referenced and images are used well with content. “File:Development of Kidney.jpg” seems an image was uploaded incorrectly, redo using same process for the rest of the images. Abnormalities are very detailed for the two completed parts, captioning of images good. Removal or completion of Horseshoe kidney is needed. Overall an informative and well-structured page, references have to properly integrated from the ureter and abnormalities sections, completion of historical findings section is also required.
Group 3 GIT
Good introduction, initial description of fore/mid/hindgut with listing of respective structures gives the reader an anatomical starting point. Fetal development is presented in appropriate depth. There is no acknowledgement of embryonic origin, research or abnormalities. These sections should feature in the introduction to present all parts of the report in the intro. The three separate timelines defeat the purpose of a timeline. These should either be merged into a single large timeline and remain at their present location or moved to introduce the foregut/midget/hindgut sections later on. Recent findings has a single study which is covered in good detail though 2-3 more studies would allow the reader to further understand current GIT Research.
In foregut section the dot-points used should match your subheadings. For example duodenum development is covered in the stomach section but is not mentioned in the subheading resulting in its development being hard to find without trawling through the text or “Ctrl-f”. Additionally you seemed to have missed out on pancreas development entirely. Foregut could also use some more images 2-3 would be suffice. Midgut development has great information, strong table, 8/8 drawings(captions required though). Inclusion of histological features gives viewer a microscopic perspective on development. Hindgut cloaca partitioning content is well worded though references are lacking.
Anorectal deformities sections should be moved under the Deformities section. The type of dotpoint style used should be standardised. Too few abnormalities in the deformities section, though after the hindgut deformities are mover there should be sufficient. There are no references supporting the possible causes of Gastroschisis. The referencing it very good unlike other pages there are no random reference subheadings. In overview format wise quite attractive, information is adequately in-depth in all sections, introduction fails to address whole page, referencing is great for a draft (exceptions being “introduction” “Liver, Gallbladder and Bile Duct”), some captions aren’t present, abnormalities in development section should be moved into deformities/abnormalities section.
Group 4 Reproductive
The Introduction, Current Models and Current Research section all in dot-point form, which obviously allowed you to more easily, put information on the page. These need to be converted into paragraph form to give the content greater readability and flow.
Presuming the system development is supposed to be the introduction, there should be inclusion of current research, historical research and abnormalities. Without these the reader will not know all the sections of the page after reading the introductory section, which is the intros purpose. The use of bold and capital letters is unneeded. The existence of a table is good though has a bunch of formatting and text problems (capitals, bold, captions, lack of lines). “(around week 4-6) that sexual differentiation occurs in the fetus ” this statement is incorrect since it is an embryo during the week4-6, it becomes a later around week 10. “450px” has not been inserted properly, the sexual differentiation image requires caption and references.
Current Research and Models has in-depth content for undifferentiated and male, though limited information on current female genital research. The headings are repetitive also many without any content, similar content needs to be merged under single headings. In Historic findings the content and wording is good but same trend continues significantly more text on Male development compared to female.
Abnormalities section is great with even attention given to female, male and both. Information is appropriately in-depth and referenced, addressing causes, process and treatment. Addition of 1-2 images in the “both” section is advised, to allow readers to identify clinical features of the diseases. Like the use of drawings especially “Abnormalities of the Uterus and Vagina” and “Anat of Testes”, you should change the caption of the testes drawing from “alt text”.
For improvement; covert of dot-points into paragraphs, expand on female sections of “current research” and “historical research”, fix a few image problems and remove unnecessary bold/capitals/captions.
Group 5 Integumentary
Introduction is short though luckily few words can go a long way, with all sections of the wiki page being properly addressed. Development overview content is good; the use of table integrated with pictures allows viewers to visually grasp the progression in skin and teeth development. The changing between dot-points and paragraph format should be standardized or use paragraphs with dot-point only for list based information. References need to be properly integrated into the page, instead of at the bottom of each section.
In the recent findings section 2 out of the 4 studies presented have any content. To improve cutting some of excess information for the 2 studies already addressed and creating summaries for the other 2 will create better scope of recent findings. The formatting of recent findings is unusual, proper placement of the “Hematoxylin/eosin” image”, removal of dot-point and removal of purple highlighting, will make the section easier for viewers to understand. Historical findings okay, more detail could be added to “skin”, “glands”, “nails”. Use of capital letters like “DEVELOPMENT” show be replaced with subheadings, the image “File:Screen Shot 2014-10-08 at 10.38.04 am.png” has not appeared properly, should be easy to fix.
Straight up the abnormalities section is amazing, no improvement needed. All 4 diseases have in-depth relevant information, sufficient referencing and images to allow viewers to visualise clinical manifestation. To improve use of dot-points or paragraphs should be standard throughout the project, referencing in beginning sections needs to compiled at ending of each Main heading or bottom of page, recent findings need 1-2 more studies, and recent findings need significant reformatting.
Group 6 Endocrine
This project would greatly benefit from an introduction, to present the contents of the project. The content is broken up into individual organs, of all the systems endocrine definitely one of the most varied in location so this approach does have some merit for initial data gathering. It does present the problem for viewers in navigation and resulting in continual repletion of timelines. Each organ is subdivided into timeline, introduction, structure, function, development and abnormalities. The content presented is solid and obviously well referenced. Placenta section should be added, because of its significant endocrine organ during fetal development. The references are mostly placed at the bottom of each organs section, these should be moved to bottom of the whole project in combination with in text citation. This will make the body of the project less cluttered and more fluid.
The table for hypothalamus hormones and associated abnormalities are mostly incomplete with “Example” filling many of the boxes. I would advise completion of hypothalamus hormone table and removal of associated abnormalities. In total there were only 3 images, addition of 2+ more images would help readers visualise the developmental organs. With at least one image per organ and preferably an additional image for an abnormality. Sufficient content is presented in this project though significant formatting changes are needed to create a completed project, additional images would be preferable.
Group 7 Neural
Introduction is a bit too focussed on anatomical division of brain and spinal cord, doesn’t address current/historical research or abnormalities. Change the word “website” to page or project. The embryonic development is explained well and should have its own subheading. Referencing is required for the whole of the introduction, Brain development and abnormalities sections. The Sections 1.1 through to 1.5 could be subheadings under the “system development” main heading.
Image showing fetal development timeline is quite overly complex and captioning/referencing is required. Additional explanation of the timeline image narrowing the viewer’s focus onto key fetal development processes will provide greater clarity allowing them to relate the image to surrounding content. The 3 images included in the project also are absent of referencing/captions present on the project page, though info after clicking on the image is great. Brain development information is in-depth and well presented(great use of table), the Spinal Cord and Meninges section have content yet to be added. There is an unusual caption  at the end of brain development which should be removed. Like the focus on anatomical structures.
There is an absence of historical research which could be found searching on Pub Med for studies completed before a certain date(1970 for example). Current Research content is sufficient. Removal of the different types of dot-points, with addition of images would make the section more attractive.Abnormalities content is great for sections covered, removal of surplus abnormalities and “500px” at the bottom of the section is need unless they are still to be added to.
--Mark Hill Succinct reviews of the projects with constructive comments. (10/10)
Lab Task 10
Taste Bud Development in Zebrafish, Danio rerio
Taste buds are chemo-sensory organs present in mouth cavity, orophayngeal cavity and lips. It is made up of modified epithelial cells which relay the chemo-sensory information to the Central nervous system. Their function is to determine whether liquids or food are edible. The study utilized a range of microscopy(light, transmission electron and scanning electron) to observe the taste bud development.
Embryo, larvae, juvenile and adult Zebra Danios (Danio rerio) were arranged according to age. Total range was 48 hours post-fertilization to 2 year old adults.
Some of the zebra fish tissue were immersed in 5% glutaraldehyde and sodium phosphate buffer for 6-10 hours. Specimens were then fixed in 1%osmium tetroxide.
- One micron sections were stained with toluidine blue and examined using light microscopy
- Ultra thin (gold and silver) sections were treated with uranyl acetate and lead citrate then examined using a Phillips or Leo Transmission electron Microscope
The rest were immersed in 5% glutaraldehyede and a phosphate buffer, then dehydrated with acetone and critical-point-dried in CO2. Specimens were coated in gold and examined using CAMscan scanning electron microscope.
In larvae 4 to 5 days old open receptor areas begin to develop in a random distribution on the lips distributed in a random manner and evenly as little hillocks on the gill arches. This is quite delayed compared to the olfactory system which receptor cells begin to appear within 30 hours of fertilization. The open receptor areas on the gill arches will later form gill rakers which taste-buds will persist on the distal region. Barbells don't develop until the juvenile period, and thus not an embryonic or fetal process.
The Scanning electron microscope showed 2 pathways taste buds can develop; it can develop at the junction of three epithelial cells and never protrude or from junction of two epithelial cells, these are associated with the border of the receptor area border. The two junction pathway push up through the apical epithelium adjacent to receptor areas creating a small hillock. Further epithelial cells contribute to the hillock, covering the top of the Tastbe bud. These early epithelial cells have a particularly prominent nucleus. Dark cells appear around open receptor areas closely followed by light cells.These cells differ in electron density(colour) and morphology of microvilli; dark cell 0.1µm wide and 0.2µm long, white cell 0.3-0.4µm wide and 0.7-0.9µm long. These micro villi give the taste buds during later development a brush like appearance
--Mark Hill This is a good summary, you are still formatting the reference link incorrectly so it appears as a number rather than showing the full reference. (5/5)
Yang, J., Li, Y., Erol, D., Wu, W., Tsai, Y., Li, X., Davis, R. and Tsang, S. (2014). Generation of induced pluripotent stem cells from conjunctiva. Graefe's Archive for Clinical and Experimental Ophthalmology, 252(3), pp.423--431.
This study aims to find if conjuntiva cells could be reprogrammed to form induced pluripotent stems, thus supplying doctors and researchers and alternative stem cell source. Presently induced pluripotent stem cells are a usueful alternative to the use of embryonic stem cells.
Method; R26(rtTa);Collal(4F2A) mice were the chosen research model. THier genomes contained four reprogramming genes Oct4, Sox2, Klf4 and Myc. Anesthetized mice had subconjunctival infection of PBS to separate conjunctival tissue. 2x3mm samples of conjuntiva was extracted via biopsy. Samples were cultured in DMEM maintaining 1ml tissue culture medium for initial 6 hours, after which 3ml of tissue culture medium was added and left for 7 days. From this iPS cell were induced and purified and spread on dishes to incubate at 37C for 12 days to reprogramming and colony isolation. COlonies were split at a 1:6 every 2-3 days.
ES cells, mouse background and conjunctiva-iPS cells were stained with Anti-SSEAI, anti-SOX2 and anti-OCT$ immunofluorescene staining. This was to examine stem cells degree of differentiation. RNA from iPS cells, and embryonic stem cells underwent PCR using specific primers. Two on the iPS cells were halted in metaphase and cultured for 2 hours, fixed in cold methanol to be anaylised under a light microscope for karyotyping
- In conjuctiva cells CK15 reactive cells increased after collegenase IV treatment
- Dox stimulated iPS cell differentiation, creating typical iPS dome colonies, by day 20 ES-like colonies had formed
- Colonised were unstainable before day 25, colonies after day 25 were strainable
- iPS stain positive for OCT4, SOX2, SSEA1, PCR also showed the expression of these genes
- iPS cells readily formed embryoid bodies, and displayed strong suppression of pluripotency gene NANOG
Discussion; Displays some potential for use of conjunctiva as a stem cell source. PCR displayed presences of pluripotency-associated transcription factors identical to those found in ES lines. This is the first time iPS has been succesfully isolated from origin conjunctiva tissue, though it is still not clear wether the iPS retain some of it original donor tissue qualities. AMD patients and diabetic patients requiring opthalmic procedures are the most viable for conjunctival tissue sampling.
--Mark Hill This is far better than your earlier stem cell review. (5/5)
TEST AREA FOR PROJECT