Talk:2009 Lab 10
Stem Cells
--Antonio Lee 15:22, 31 October 2009 (EST) Hi Mark, I have marked the Lab10 Questions and indicated marks on students' pages. Thanks for the opportunity, I enjoyed it very much!
Practical Preparation
--Antonio Lee 10:33, 8 October 2009 (EST) Hi Mark, I have uploaded Lab 10 Assessment Questions on both the Lab Page and Student's Page. I will make myself down to the lab at 12:45-50 this afternoon. Thanks.
--Antonio Lee 11:26, 2 October 2009 (EST) Hi Mark, I have updated each group discussion boards with assigned links and PDFs (uploaded). Please take a look and let me know if they are okay. I will put couple of assessment questions before the lab next week. Thanks,
--Antonio Lee 09:22, 2 October 2009 (EST):Hi Mark, Yes, I'd definitely like to provide the readings beforehand and encourage them to read their paper beforehand. I will try to paste each paper on assigned group's discussion board. I am sure not all students will read their papers before hand and I appreciate during 15-20 minutes it is impossible to digest the full content of the paper. That is why I thought each student from the group (of 4 students) can address 1 question only during the class time (essentially, Intro, M&M, Results or Discussion). Then each student can present their bits in couple of minutes to make up the whole story the paper is saying?
- I will also make sure to leave ~15 mins at the end for them. Thanks,
--Mark Hill 06:57, 2 October 2009 (EST) Hi Antonio, looks good so far. I have not yet checked the length of your readings for the group work, but you may consider how long it will take them to do this. Do you want to provide the readings to the groups beforehand? If so, you can paste it onto each group discussion page and see whether some read the paper before the lab (some listed papers are not accessible from outside UNSW). Also, the students will require some time (15 minutes or so) at the end of the lab to discuss their ongoing group project.
--Antonio Lee 21:13, 1 October 2009 (EST) Hello Mark, I have put the materials on the Lab Page. Could you take a look and let me know if the format/use of external links are appropriate? I will also set 2-3 easy questions and post them on the page for the students before the lab next week. Thanks,
--Mark Hill 11:48, 18 September 2009 (EST) I now have permission for this image if you want to use the mitochondrial example. Swapping_mitochondrial_DNA_mammalian_oocytes
--Antonio Lee 10:34, 18 September 2009 (EST)Thanks Mark, just trying to get used to in using this Wiki - it's my first. I take all of your comments on board and will work on those over the weekend. Thanks.
--Mark Hill 09:56, 18 September 2009 (EST) Yes I do fine email back n forth a little tedious. Lets work together here. Your class plan looks fine to me so far.
Title: Therapeutic Use of Stem Cells - Practical Hurdles & Ethical Issues
- --Mark Hill 10:10, 18 September 2009 (EST) I have now renamed the actual practical page to this title. Feel free to change any content yourself. I think the students also need a good but brief description of new terms you will be using in your presentation. See example Muscle Lab 7 Terms.
Structure
- --Mark Hill 09:59, 18 September 2009 (EST) These don't have to be power points, but if you do use, need to convert to PDF and link to the lab page. I usually prepare 2 PDF versions (1 slide /page and 4 slides / page printing)
- 15 min: (PPT) Brief introduction on Embyonic and Adult Stem cells (I will borrow some of your slides and incorporate my own) & their potentials as new therapeutic agents.
- 15 min: (PPT) Outline our own work - Developing better stem cell transplantation strategy using chemotherapy & genetically engineered chemo-resistant stem cells for treating muscle diseases (muscular dystrophies).
- --Mark Hill 10:03, 18 September 2009 (EST) You might also consider using this very recent example for Mitochondrial Disease as well, because its very current, therapeutically relevant and just interesting new application, and the paper in nature also has an illustrated editorial. PMID: 19710649 | Nature Article | Nature editorial
- 20 min: (Web Search Exercise) Each group of 4 students (pre-existing groups) will be given a recent news article on stem cell break-through / advancement with clinical/therapeutic implication (I will pre-select these from news feeds and post them on the web-site 1 week before the lab, one assigned to each group). They will do web-search exercise to prepare answers / comments for each of the following questions regarding their news piece.
Q1. What is the background to the existing problem / disease condition?
Q2. What approach / method did the research team take to tackle / improve the problem?
Q3. What was the breakthrough / major advancement OR failure / drawback? and why might this be of significance?
Q4. What are the next steps in moving forward? What are the next or new hurdles to overcome?
Q5. Are there any ethical / moral issues or concerns in your view?
Suggestive format will be that each of the 4 students will prepare a paragraph-long answer to 1 of 4 questions (from Q1 ~ Q4) using web-search. Each student then can express their view on Q5 verbally during discussion. They can type-up the answers as they go (or copy and paste relevant information from web-pages and include the http address for reference).
50-60 min: (Student Participation) Each group will present their answers (5 min) followed by questions/discussions (5 min).
I will have prepared my own version of "answers" for each of the news piece to facilitate.
If you require some sort of assessable material, how about if we ask the students to hand-in the write-up of their answers (Q1 ~ 4), say by the following Thursday? - to give them little more time to polish their paragraphs?
- --Mark Hill 11:50, 18 September 2009 (EST) I have only been setting relatively easy questions to mark as part of their ongoing assessment. ANAT2341_2009_Students#Progressive_Assessment
That is all, and by all means, if you find making comments on e-mail laborious, do drop by (or I can come down to your office) and we can have a chat.
Previous Webpage
- --Mark Hill 11:44, 18 September 2009 (EST) Feel free to use what you need from below.
The term "stem cell" is used so freely these days in many different forums that it is difficult sometimes understand without context what scientists, politicians, ethicists and commentators are discussing. This lecture will focus on the cell biology of stem cells and the current research on growing and differentiating theses cells.
Background information can also be found at UNSW Embryology Stem Cells and Week 1 Development.
Why are they in the News?
- Scientific and Ethical
- Therapeutic uses
- Issues relating to human cloning
- Use of excess human eggs/sperm for research purposes
- Availability of human stem cell lines
What can they be used for?
- Generation of “knock out” mice
- Studying regulation of cell differentiation in development
- Therapeutic uses?
- Genetic disease
- Neurodegenerative
- Injury
PubMed
- Medline Search “stem cell”
- 2002 - 110,920
- 2004 - 128,485
- 2005 - 140,966
- 2006 - 154,176
Research that led to Stem Cells
- Human Diseases
- Generation of “knock out” mice
- Human Development
- Studying regulation of cell differentiation in development
- Human Reproduction
- Disorders, sterility
Tissue Stem Cells
- differentiated cells have short life spans continually replaced
- blood cells, epithelial cells of skin and digestive tract
- fully differentiated cells do not proliferate
- proliferation of less differentiated- stem cells
- produce daughter cells that either differentiate or remain as stem cells
Blood Cells
- All different types of blood cells develop from a pluripotent stem cell in bone marrow
- Precursors of differentiated cells undergo several rounds of cell division as they mature
- proliferation ceases at terminal stages of differentiation
Embryonic Stem Cells
NIH - What are embryonic stem cells?
Pluripotent Stem Cells
- What is a stem cell- Pluripotent
- Pluripotent - to describe stem cells that can give rise to cells derived from all 3 embryonic germ layers
- Mesoderm
- Endoderm
- Ectoderm
- layers are embryonic source of all cells of the body
Blastocyst
- hollow structure composed of about 100 cells surrounding an inner cavity
- Only ES cells, which form inner cell mass, actually form the embryo.
- ES cells can be removed from the blastocyst and grown on lethally irradiated “feeder cells.” (See E. Robertson et al., 1986, Nature 323:445)
Stem Cell Definition
- cell that has ability to divide for indefinite periods
- self replicate
- throughout life of organism
- stem cells can differentiate
- conditions, signals
- to the many different cell types
Chimeric Mouse
- ES or teratocarcinoma
- shows that stem cells can combine with cells of a normal blastocyst to form a healthy chimeric mouse
Embryoid Bodies
- spheroid cellular tissue culture structure
- mouse and human ES cells have the capacity to undergo controlled differentiation
- recapitulate some aspects of early development
- regional-specific differentiation program
- derivatives of all three embryonic germ layers
Historic References
Mouse
- Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Martin GR. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7634-8.
- Characterization of a pluripotent stem cell line derived from a mouse embryo. Wobus AM, Holzhausen H, Jakel P, Schoneich J. Exp Cell Res. 1984 May;152(1):212-9.
- Transgenesis by means of blastocyst-derived embryonic stem cell lines Proc Natl Acad Sci U S A. 1986 Dec;83(23):9065-9. Gossler A, Doetschman T, Korn R, Serfling E, Kemler R.
Pig and Sheep
Derivation of pluripotent, embryonic cell lines from the pig and sheep. Notarianni E, Galli C, Laurie S, Moor RM, Evans MJ. J Reprod Fertil Suppl. 1991;43:255-60.
Primate
Isolation of a primate embryonic stem cell line. Thomson JA, Kalishman J, Golos TG, Durning M, Harris CP, Becker RA, Hearn JP. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7844-8.
Human
Embryonic stem cell lines derived from human blastocysts. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Science. 1998 Nov 6;282(5391):1145-7.
Stem Cell Lines ATCC - Embryonic Stem cell lines
Cord Blood Stem Cells
- Blood collected from the placental umbilical cord of a newborn baby shortly after birth
- total amount of blood about 90 ml
- blood stem cells that can be used to generate red blood cells and cells of the immune system
- collected, typed, stored in Cord Blood Bank
- Both public and private Banks have arisen
- available for use by the donor and compatible siblings
- suggested use to treat a range of blood disorders and immune system conditions such as leukaemia, anaemia and autoimmune diseases
- cells provide a resource for bone marrow replacement therapy in many diseases
Cord Blood - Disease Treatments
- Acute Lymphoblastic Leukaemia
- Acute Myeloblastic Leukaemia
- Adrenoleukodystrophy
- Blackfan-Diamond
- Chronic Myeloid Leukaemia
- Chronic Lymphocytic leukaemia
- Fanconi's Anaemia
- Hurler's Syndrome
- Krabbe's disease
- Lymphomas
- Myelodysplastic Syndrome
- Mucolipopolysaccharide deficiency
- Osteopetrosis
- Syndrome Severe Aplastic Anaemia
- Severe Combined Immunodeficiency Disease
- Thalassaemia
- Wiskott-Aldrich Syndrome
- Miscellaneous
- Cancer
- Genetic disorders
- Immune deficiency
- Storage disorders
Adult Stem Cells
NIH - What are adult stem cells?
Stem Cells in the Adult
- Connective Tissue
- Bone marrow
- Blood Cells, Osteoclasts, blasts
- Epithelia
- Gut
- Skin
- Neural?
Epidermis: Immortal Stem Cell
Induced Pluripotent Cells
- non-pluripotent cells engineered to become pluripotent
- a cell with a specialized function ‘reprogrammed’ to an unspecialized state
Stem Cell Markers
In order to carry out research on stem cells, it is important to be able to identify them. A number of different research groups in the late 90's generated several antibodies which specifically identified undifferentiated, differentiating or differentiated stem cells from a number of different sources and species. Note that the nomenclature in some cases is based upon the antibody used to identify the cell surface marker.
- Every cell surface has specialized proteins (receptors) that can selectively bind or adhere to other “signalling” molecules (ligands)
- Different types of receptors differ in structure and affinity for signalling molecules
- Cells use these receptors and molecules that bind to them as a way of communicating with other cells and to carry out their proper functions in the body
- Stage-Specific Embryonic Antigen-1 (SSEA-1) cell surface embryonic antigen which has a role in cell adhesion, migration and differentiation and is often differentially expressed during development. Can be identified by Davor Solter monoclonal antibody MC-480 (SSEA-1).
- Stage-Specific Embryonic Antigen-4 (SSEA-4) cell surface embryonic antigen of human teratocarcinoma stem cells (EC), human embryonic germ cells (EG) and human embryonic stem cells (ES) which is down-regulated following differentiation of human EC cells. Antigen not expressed on undifferentiated murine EC, ES and EG cells but upregulated on differentiation of murine EC and ES cells. Can be identified by Davor Solter monoclonal antibody MC-813-70 (SSEA-4)
- Tumor Rejection Antigen (TRA-1-60) Sialylated Keratan Sulfate Proteoglycan expressed on the surface of human teratocarcinoma stem cells (EC), human embryonic germ cells (EG) and human embryonic stem cells (ES).
- Tumor Rejection Antigen (TRA-1-81) antigen expressed on the surface of human teratocarcinoma stem cells (EC), human embryonic germ cells (EG) and human embryonic stem cells (ES).
- Both TRA antibodies identify a major polypeptide (Mr 240 kDa) and a minor polypeptide (Mr 415 kDa).
- Oct-4 (Pou5f1 – Mouse Genome Informatics) gene has an essential role in control of developmental pluripotency (Oct4 knockout embryo blastocysts die at the time of implantation). Oct4 also has a role in maintaining viability of mammalian germline.
- Stem Cell Antigen 1 (Sca-1) member of the Ly-6 family of GPI-linked surface proteins (Mr 18 kDa) and a major phenotypic marker for mouse hematopoietic progenitor/stem cell subset.
- CD133, AC133, prominin 5 transmembrane glycoprotein (865 aa) expressed on stem cells with hematopoietic and nonhematopoietic differentiation potential.
- Alkaline Phosphatase
- embryonic stem cell is characterized by high level of expression alkaline phosphatase (undifferentiated state) ATCC ELF Phosphatase Detection Kit for Embryonic Stem Cells
- assay to determine if embryonic stem cells are undifferentiated or are starting to differentiate
- uses a fluorescent detection of endogenous phosphatase activity in embryonic stem cells
PNAS - Expression of molecular markers characteristic of ES cells in morula-derived cell lines
Stem Differentiation
Epithelium
- each generation at least 1 "immortal" stem cell
- descendants present in patch in future
- Other basal cells
- leave basal layer and differentiate
- Committed, born different
or may be stem cells equivalent to immortal stem cell in character mortal in sense that their progeny jostled out of basal layer and shed from skin
Amplifying Cells
- Stem cells in many tissues divide only rarely
- give rise to transit amplifying cells
- daughters committed to differentiation that go through a limited series of more rapid divisions before completing the process.
- each stem cell division gives rise in this way to eight terminally differentiated progeny
Stem Cell Production - Stem Cell Daughter Fates
- Environmental asymmetry
- daughters are initially similar
- different pathways according to environmental influences that act on them after they are born
- number of stem cells can be increased or reduced to fit niche available
- Divisional asymmetry
- stem cell has an internal asymmetry
- divides in such a way two daughters are already have different determinants at time of their birth
