Ipsc have made a lot of achievement but it was hard to show all of them so I have selected only the very important onesMouse-> skin cardiomyocyte even sperm produced through this tech !Human-> fibroblast blah blahblah
WASHINGTON (Reuters) - In a surprise result that can help in the understanding of both aging and cancer, researchers working with an engineered type of stem cell said they reversed the aging process in a rare genetic disease.In this case, they wanted to study a rare, inherited premature aging disorder called dyskeratosis congenita. The blood marrow disorder resembles the better-known aging disease progeria and causes premature graying, warped fingernails and other symptoms as well as a high risk of cancer.It is very rare and normally diagnosed between the ages of 10 and 30. About half of patients have bone marrow failure, which means their bone marrow stops making blood and immune cells properly.But, reporting in Thursday's issue of the journal Nature, they said the process of making the iPS cells appeared to reverse one of the key symptoms of the disease in the cells.In this disease, the cells lose telomerase, an enzyme that helps maintain the telomeres. These are the little caps on the ends of the chromosomes that carry the DNA.A gene called TERC helps restore the telomeres and Daley's team said it may be that tumor cells make use of TERC to become immortal.In making the iPS cells and getting them to grow in the lab, Daley's team discovered they had three times as much TERC as the diseased cells they were made from.Simply turning the skin cells into iPS cells helped restore their damaged telomeres, Daley's team reported. This in theory stops a major component of the aging process as well."We're not saying we've found the fountain of youth, but the process of creating iPS cells recapitulates some of the biology that our species uses to rejuvenate itself in each generation," Daley's colleague Suneet Agarwal said in a statement.
Introduction Stem Cells Ability to self renew, to makeexact copies of itself through celldivision. Ability to develop into differenttypes of mature cells. Image adopted from:- http://clearlyexplained.com/nature/life/cells/stemcelltree.gif
Stem Cells Have the ability to specialize in all postembryonic tissues and organs, and also extra embryonic tissues. E.g.- zygote Capable of giving rise to most tissues of an organism. E.g.- ES cells MultipotentStem cells that can differentiate into many family of Can only differentiate into single type of cell cells, E.g.– Hematopoietic stem cells but have the property of self-renewal, E.g.- mucus membrane cells and skin stem cell Adopted from:- http://www.wikipedia.org
Induced pluripotent stem cells (iPSCs) aretypically adult cells that have beengenetically reprogrammed to an embryonicstem cell–like state by being forced toexpress genes and factors important formaintaining the defining properties ofembryonic stem cells.
iPSc-made from mouse-> leadto the creation of beating cardiomyocyte, skinand almost every kind of cell you canimagine.iPSc made from human-> alsolead to the creation of various cell types inbody, in addition even produced gamete!iPSc created live organism->“ Xiao Xiao / Tiny”
In June 2007 Yamanaka et al, Kyoto University & two other independent groups Harvard, MIT & UC, Los Angeles showed successful reprogramming and production of viable chimeras Instead of Fbx15 they used Nanog as a marker of pluripotency detection DNA methylation patterns were identical to ESCs
iPSc-made from mouse-> lead tothe creation of beating cardiomyocyte, skinand almost every kind of cell you canimagine.iPSc made from human-> alsolead to the creation of various cell typesin body, in addition even producedgamete!iPSc created live organism->“ Xiao Xiao / Tiny”!
For humans the same procedure was followed, exceptingthe addition and substitution of a few other factors like:- OCT 4, SOX 2, NANOG, LIN 28 genes & lentiviral system- by Thompson J. POU5F1(OCT4), C-MYC, KLF4, SOX2 genes & retroviruses- by Yamanaka S. PCM Reprogramming factors FM Fibroblasts iPSC (NANOG-GFP) Adopted from:- http://www.systembio.com/downloads/StemCellposter.pdf
iPSc-made from mouse-> lead tothe creation of beating cardiomyocyte, skinand almost every kind of cell you canimagine.iPSc made from human-> alsolead to the creation of various cell typesin body, in addition even producedgamete!iPSc created live organism->“ Xiao Xiao / Tiny” !
ADOPTED FROM: Stem cells: The magic brew, Janet Rossant. Nature 448, 260-262 (19 July 2007); doi:10.1038/448260a
TETRAPLOID COMPLEMENTATION FORMS ONLY PLACENTA AND 2N EXTRA-EMBRYONIC MEMBRANE FUSION 4N 2NWHITE COAT COLOUR “XIAO XIAO “/ “ TINY “ / AN ALL - iPSC MOUSE SKIN REPROGRAMMING iPSC BLACK COAT CAN FORM ALL POTENTIAL COLOURBLACK COAT TYPES OF CELLS IN BODY COLOUR
Defined factors Motor neuronsAdopted from:- http://www.rndsystems.com/cb_detail_objectname_cb09i2_induced_pluripotent_stem_cells.aspx
Morphology- cell have round shape, large nucleolus and scant cytoplasm; colony morphology is also similar. Growth properties-iPSCs are mitotically active, actively self-renewing, proliferating, and dividing at a rate equal to ESCs. Pluripotent Stem cell markers:- iPSCs express cell surface antigenic markers expressed on ESCs. Pluripotent Stem Cell Genes- Oct-3/4, Sox2, Nanog, GDF3, REX1, FGF4, ESG1, DPPA2, DPPA4, and hTERT. Telomerase activity- express high telomerase activity.
Neural differentiation-βIII-tubulin, GFAP expressed (found specifically in neurons and glial cells). Cardiac differentiation- Differentiated into cardiomyocytes that spontaneously began beating. Teratoma formation- iPSCs injected into immunodeficient mice spontaneously formed teratomas after nine weeks. Embryoid body- iPSCs also form embryoid bodies and have peripheral differentiated cells. Chimeric mice-Mice with iPSC derivatives incorporated all across their bodies with 10%-90% chimerism. Tetraploid complementation-Whole, non-chimeric, fertile mice produced, although with low success rate.
Promoter demethylation- Promoters of pluripotency-associated genes, including Oct-3/4, Rex1, and Nanog, were demethylated in iPSCs, demonstrating their promoter activity. Histone demethylation-H3 histones associated with Oct-3/4, Sox2, and Nanog were demethylated.
HbS /HbS HbS /HbS HbA /HbSHbS /HbS Scientists of MIT, have used iPS cells to cure sickle cell anemia. Adopted from:- 21 DECEMBER 2007 VOL 318 SCIENCE www.sciencemag.org
Whitehead Institute researchers rescued Parkinsons phenotype in rat, transplanting committed neurons free of contaminating undifferentiated cell population & thus minimizing risk of tumor formation. Reprogramming Redifferentiation Transplantation into adult brain of parkinson’s disease rat model,after removal of contaminating pluripotent cells from committed neurons using FACS
Whitehead Institute researchers produced Parkinson’s disease patient-specific stem cells free of harmful reprogramming transgenes Skin cells of parkinson’s patient Retroviruses packaged with reprogramming genes flanked by lox-p sequence iPS cells with integrated transgenes Retroviruses packaged with cre enzyme iPS cells without transgenes Differentiatng factors Dopaminergic neurons Stain Green-> class III beta-tubulin (neuron specific) Red-> tyrosine hydroxylase (dopaminergic neuron specific) Adopted from:- http://www.wi.mit.edu/news/archives/2009/rj_0305.html
Suneet Agarwal & George Daley from Childrens Hospital Boston and the Harvard Stem Cell Institute were studying Inherited premature aging disorder- Dyskeratosis congenita A progeroid disease Bone marrow failure Cells lose telomerase activity due to reduced expression of TERC(Telomerase RNA Component) BUT iPSC from skin of such patients shows TERC expression three times more than diseased cell Thus simply turning the diseased skin cells into iPS cells helped restore their damaged telomeres at normal level Published online in NATURE 17/2/2010
iPS Cells can be used for toxicological studies thatcould save lives & money as these iPSC derivedtissues can be exposed to a vast library of chemicalcompounds to test them as potential drugs. Human disease can be studied now in humantissues i.e. worth studying in mouse models increasingthe level of accuracy. Making iPSC showed the link between stem cellsand cancer cells, as the reprogramming factors are ingeneral cancer critical genes (c-myc, klf4 and others).
No transplant rejectionNo ethical issues involved as nousage of embryosAs a method of cloning, the nonselfcytoplasmic inheritance is absenthere unlike SCNT
Oncogenic effect of retroviral integrationDue to c-myc 20% chimeric mice developed cancerForced reprogramming related safety issuesTeratoma related safety issuesRe-differentiation related safety issue
Trans-differentiation is the process when a differentiatedcell creates cells outside its established differentiationpath i.e. the cell fate switches, including theinterconversion of stem cells Trans-differentiation was shown earlier by convertingpancreatic exocrine cell to beta cell But now neurons are derived from fibroblasts !
Marius Wernig et al.demonstrated thatfibroblasts convert to functional neurons in vitro by forced expression of three factors:- Ascl1, Brn2, and Myt1l, bypassing the pluripotent stage, thus avoiding the risk of tumor formation. ? •MAP2, NeuN, and synapsin positive, •Produces action Brn2, Myt1l & Ascl1 potentials 12 days mEF iNs Nature. Published online January 27, 2010. doi:10.1038/nature08797
Instead of using retroviruses, researchers have tried using: Plasmid expression vector- Yamanaka S. et al ADVANTAGE- no evidence of transgene integration, no risk of transformation due to integration. DISADVANTAGE- low efficiency Adenoviral vectors- Konrad H. et al ADVANTAGE- no evidence of transgene integration, no risk of transformation due to integration. DISADVANTAGE- products get diluted out during successive cell divisions in progeny cells; efficiency poor Proteins channelized to cell via poly arginine anchors- Ding S. et al ADVANTAGE- no evidence of transgene integration, no risk of transformation due to integration. DISADVANTAGE- products get diluted out during successive cell divisions in progeny cells; efficiency poorc-MYC was avoided due to its proto-oncogenic nature- Yamanaka S. et al the cells took longer time to become iPSCs; low efficiency
What are the exact sequence of molecular events that leads to this dramaticreprogramming?Whether additional changes, beyond the expression of the four transcriptionfactors, are involved or not?The process of reprogramming is slow — colonies take up to 20 days todevelop into real ES-like cells, and their frequency is quite low. Is this because only a few cells happen to express the right combination orlevels of the four factors because of the random integrations of theretroviruses? Or, are there additional events, perhaps associated with retroviral insertion,that are required for full transformation?
“This will be the long-term solution”- SirMartin Evans“This is the future of stem cell research. Itshundered times more interesting thanSCNT”- Dr. Ian Wilmut“A decade from now, this (hESC)controversy will be just a funny historicalfootnote.” – Dr. James Thompson.“Embryonic stem cells are not safe, but atthe moment, iPS cells are moredangerous.” – Shinya Yamanaka
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