lecture 29: stem and iPS cells Flashcards

Question

What are iPSCs?

Answer 1

* induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors * question: ESC factors responsible for reprogramming, but which ones? * No drug-resistant colonies obtained with any single factor * all 24 factors → 22 resistant colonies * 12 continued, 5 exhibited ESC morphology * stepwise elimination of each factor to establish the 4 critical factors sufficient to form iPS cells

Answer 2

* virion * fusion * uncoating and reverse transcription * incorporation of cellular proteins * intracellular trafficking * nuclear entry * access to chromatin * integration

Answer 3

* no drug-resistant colonies obtained with any single factor * all 24 factors → 22 resistant colonies * 12 continued, 5 exhibited ESC morphology * epigenetic markers and gene expression of key regulatory factors were not comparable * 4 critical factors were necessary and sufficient for the formation of iPS cells: * cMyc * Klf4 * Sox2 * Oct4 * improved colony formation with selected factors

Answer 4

* iPS contribute to all germ layers in vitro and express ESC markers * but retain somatic memory

Answer 5

* yes * induction of pluripotent stem cells from adult human fibroblasts by defined factors

Answer 6

* add Oct4, Sox2, cMyc and Klf4 to somatic cells * intermediate cells (transient population) * somatic markers silenced * activation of SSEA1 * partially reprogrammed cells * stable cell lines * viral transgenes on * proliferation genes activated * pluripotency genes silent * aberrant expression of lineage genes * teratomas, but no adult chimeras * iPS cells * silencing of retroviral transgenes * activation of pluripotency genes * activation of telomerase * reactivation of silent X chromosome in female cells * teratomas and germline chimeras * knockdown of lineage genes * inhibition of DNA methylation * evolution of factor delivery

Answer 7

* mRNA expression * early-passage iPSCs are distinct from ESCs, reflecting expression from the cell of origin * late-passage iPSCs are nearly identical to ESCs * miRNA expression * the imprinted Dlk1-Dio3 cluster is not expressed in most iPSC lines * IncRNA expression * not determined * histone modifications * those modifications tested (H3K4me3 and H3K27me3) seem to be indistinguishable between ESCs and iPSCs * DNA methylation * distinct at early passage, reflecting the pattern of target cells * late-passage cells are nearly identical * X chromosome activation status * both iPSCs and ESCs are XaXa * metabolism * not determined

Answer 8

* mRNA expression * early passage iPSCs are distinct from ESCs reflecting expression from the target cell * late passage iPSCs are closer to ESCs * miRNA expression * some differences have been described * but no consistent differences have been found across multiple ESC and iPSC lines * IncRNA expression * differences have been described * some have functional roles in reprogramming * histone modifications * two modifications (H3K4me3 and H3K27me3) seem to be identical * H3K9me3 is different * DNA methylation * some differences have been described * X chromosome activation status * human ESCs are mostly XiXa but can be XaXa depending on culture condition * human iPSCs are XaXi * metabolism * identical or nearly identical

Answer 9

* human embryonic stem cells derived by somatic cell nuclear transger * comparison of mouse iPS cells with SCNT-ESCs and ESCs suggest that SCNT-ESCs are subtly closer to ESCs, as defined by methylation marks and differentiation capacity * oocyte cytoplasm reprogrammes the somatic nucleus in a different manner to OSKM → will help ID novel factors * creates a method to eliminate a small proportion of mitochondrial diseases (debating approved in Britain for clinical application)

Answer 10

* adult stem cells * acceptable to derive * tissue specific * limited expansion in vitro * very difficult to isolate (usually a rare population) * existing human ES cell lines * very few cell lines exist * not well characterised * do not eliminate risk of immune rejection * ES cells derived from SCNT * ethical concerns with creating embryos * patient specific cell lines (avoid immune rejection) * potential exploitation of egg donation process * iPS cells * acceptable to derive * patient specific cell lines (effective) * not well characterised * may not be fully reprogrammable

Answer 11

* iPS Cells derived from somatic cells of patients with genetic disease

Answer 12

* treatment of sickle cell aneamia mouse model with iPS Cells generated from autologous skin * corrected sickle cell anaemia defect * gene targeting to correct beta globin mutation * generation and differentiation of iPS * → driving potential for patient specific iPS cells

Answer 13

* human ES cells * establish pure cultures of specific cell type * lineage restriction by cell survival or cell sorting (e.g. insulin promoter driving antibiotic resistance gene or GFP) * induce with supplemental growth factor(s) or inducer cells (e.g. retinoic acid for neural cells) * test physiologic function * in vitro (e.g. stimulated insulin release) * demonstrate efficacy * in rodent models * in non-human primate model with rhesus ES cell-derived cells (e.g. diabetes and Parkinson's disease models in primates) * evaluate integration into host tissue (e.g. cardiomyocytes for treatment of heart failure) * ? recurrent autoimmunity (e.g. diabetes) * demonstrate safety * in non-human primate model with rhesus ES cell-derived tissues * show absence of tumour formation * show absence of transmission of infectious agents * test methods to prevent rejection * multi-drug immunosupression * create differentiated cells isogenic to prospective recipient using nuclear reprogramming * transduce ES cells to express recipient MHC genes * establish haematopoietic chimera and immunologic tolerance * human trials

Answer 14

* scientific basis is not always clear * pre-clinical data is not always in literature (no peer review) * good manufacturing practice ????? * methods often not disclosed * no safety data * no patient follow up * e.g. scandal-hit stem cell clinic closes * berlin * europe's largest stem cell clinic, which is at the centre of a scandal over the death of a baby given an injection to the brain, has shut * the closure of the XCell-centre in Duesseldorf follows an undercover investigation by the Sunday Telegraph into its controversial practices * the clinic, which attracted hundres of patients from Britain, charged up to 20,000 pounds for stem cell injections into the back and brain despite a lack of sceintific proof that the treatments worked * foetal precursor stem cells from certified closed colony of rabbits of 30 generations onwards, we also offer autologous precursor stem cells from pheripheral blood of patients * stem cell tourism deaths spark inquiry * foetal stem cell injections create brain tumours in isreali boy

Answer 15

* first fully synthetic organ transplant saves cancer patient

Answer 16

* each medical problem will have a preferred solution * it may be an ESC derived solution, or ASC, or a combination of both * there is a need to maintain research on the applications of both cell types * both stem cell types have advantages * both stem cell types have disadvantages * the most exciting thing: there are patients being enrolled in clinical trials for eSC derived and ASC derived therapies

Answer 17

* two defining properties of stem cells are their * ability to self renew * ability to differentiate/regenerate * embryonic stem cells are pluripotent; most adult stem cells are multipotent * self renewal is orchestrated by a complex network of intrinsic and extrinsic factors * differentiation of cells is no longer a one-way street; cell fates can be reset by epigenetic programming * efficacy, cell functionality and stability must be demonstrated before therapeutic use