lecture 29: stem and iPS cells Flashcards
1
Q
What are the different types of stem cells?
A
- Embryonic stem cells
- isolate from the inner cell mass of the blastocyst
- origin: blastocyst of embryo
- strengths:
- pluripotent (3 germ layers)
- self-renewal and high replicative capacity
- weaknesses:
- immunological concerns
- subject to ethical debate
- potential for teratoma and teratocarcinoma
- currently no clinical trial data
- Adult SCs
- origin: bone marrow, circulation or resident tissue
- strengths:
- autologous
- clinical safety and efficacy data
- typically lineage commited
- weaknesses:
- limited number
- limited replicative capacity
- lineage restricted
- iPSCs
- origin: reprogramming of somatic cells
- strengths:
- totipotent (3 germ layers and trophoblast)
- autologous
- large reservoir of cells
- weaknesses
- potential for teratoma and teratocarcinoma
- no clinical data
2
Q
What defines a stem cell?
A
- self-renewal - maintenance of ‘stemness’
- potency/potential - capacity for differentiation
- indefinite proliferation
- telomerase activity
- normal karyotpe maintained
- marker expression profiles
- embryoid body formation
- teratoma formation
- directed: neurons, cardiomyocutes, haematopoietic progenitors, insulin producing cells
3
Q
What are differing potencies of stem cells?
A
- totipotent: fertilised oocyte and cells after first cleavage divisions; ability to form entire organism
- pluripotent: cells of the ICM of the blastocyst; ability to form all three germ layers but not the extraembryonic tissues; embryonic stem cells
- multipotent: mesenchymal stem cells which can form bone, cartilage and fat; ability to form multiple cell types; adult stem cells
4
Q
What is embryo development?
A
- day 0: fertilisation
- fertilised egg (zygote)
- day 1: first cleavage
- day 2: 2 cell stage
- day 3 - 4: 4 cell stage, 8 cell uncompacted morula
- day 4: 8-cell compacted morula
- day 5: early blastocyst, trophectoderm, blastocoel, inner cell mass
- day 6 - 7: late-stage blastocyst, leaving zona pellucida
- day 8 - 9: implantation of the blastocyst: epiblast, hypoblast
5
Q
What are the varying tissue lineages over the course of embryo development?
A
- fertilised egg ( day 0)
- blastocyst (5)
- trophoblast (6-7)
- cytotrophoblast (8-9)
- syncytioptrophoblast (12)
- cytotrophoblast (8-9)
- inner cell mass (6-7)
- hypoblast (8-9)
- extraembryonic endoderm (12)
- yolk sac (14)
- extraembryonic endoderm (12)
- epiblast (8-9)
- amniotic ectoderm (12)
- primitive ectoderm (12)
- embryonic ectoderm (15)
- primitive streak (14)
- embryonic endoderm (15)
- embryonic mesoderm (15)
- extraembryonic mesoderm (15)
- hypoblast (8-9)
- trophoblast (6-7)
6
Q
What is the history of embryonic stem cells?
A
- establishment in culture of pluripotent cells from mouse embryo
- isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells
- isolation of a primate embryonic stem cell line
- embryonic stem cell lines derived from human blastocysts
- Odorico et al (2001):
- cleavage stage embryo
- cultured blastocyst
- isolated inner cell mass
- cultured on irradiated mouse fibroblast feeder cells
- cells dissociated and repleted
- new feeder cells
- established ES cell cultures
7
Q
What are embryonic stem cells?
A
- all human lines available are derived from excess embryos (IVF)
- process of isolation (immunosurgery, laser)
- many isolated onto mouse embryonic fibroblast (MEF) feeder layers, with (bovine) serum
- undefined conditions
- antigens found on stem cells
- disease transmission from use of animal products
- FDA will not approve use for transplantation
- late passage numbers available for study (adaptation with culture)
- enzymatic passaging protocols
- karyotypic instability
8
Q
What controls self-renewal?
A
- sox 2
- oct 4
- nanog
- klf 4
- myc
9
Q
What is a model core ES cell regulatory circuitry?
A
10
Q
What characterises embryonic stem cells?
A
- morphology
- transcription factor expression
- dependence on glycolytic metabolism and glutaminolysis
- long telomeres, high telomerase activity
11
Q
What characterises pluripotency?
A
- chimera formation
- differentiation (in vitro, teratoma formation in vivo)
12
Q
What is developmental potential/differentiation?
A
- stem cell division and differentiation
- symmetric division
- asymmetric division
- progenitor division
- terminal differentiation
- pluripotent cell → unipotent
- induction (environmental): growth factors; other factors
- commitment (cell autonomous): epigenetic; transcription factor networks
- patterning (environmental): positional information
13
Q
What is the relationship between developmental potential and epigenetic status?
A
- totipotent zygote
- global DNA demethylation
- pluripotent
- e.g. ICM ES cells, EG cells, EC cells, mGS cells, iPS cells
- 2 active X chromosomes
- global repression of differentiation genes by Polycomb proteins
- promoter hypomethylation
- multipotent
- e.g. adult stem cells (partially reprogrammed cells?)
- x inactivation
- repression of lineage-specifc genes by polycomb proteins
- promoter hypermethylation
- unipotent
- e.g. differentiatied cell types
- x-inactivation
- derepression of polycomb silenced lineage genes
- promoter hypermethylation
14
Q
What are epigenetic mechanisms?
A
- affected by these factors and processes:
- development (in utero, childhood)
- environmental chemicals
- drugs/pharmaceuticals
- ageing
- diet
- DNA methylation
- methyl group (an epigenetic factor found in some dietary sources) can tag DNA and activate or repress genes
- histones are proteins around which DNA can wind for compaction and gene regulation
- histone modification
- the binding of epigenetic factors to histone “tails” alters the extent to which DNA is wrapped around histones and the availability of genes in the DNA to be activated
- health endpoints
- cancer
- autoimmune disease
- mental disorders
- diabetes
15
Q
How does the epigenetic response to extrinsic signals occur?
A
- occurs through a network of transcription factors
- active genes
- active chromatin
- pluripotent cells
- differentiatied cells
- master regulators e.g. oct 4, NANOG, Sox 2 etc
- auxillary factors: myc etc
- lineage specific genes upon differentiation
- poised genes
- bivalent chromatin
- pluripotent cells
- genes of early response to differentiation,
- silent genes
- silent chromatin
- pluripotent cells
- differentiated cells
- lineage specific genes in ES cells
- master regulators upon differentiation
16
Q
What are adult stem cells?
A
- drive the renewal of all adult tissues
- divide continuously to produce new cells that undergo a robust differentiation programme
- limited repair and regeneration
- in culture:
- highly refractory to expansion and long-term culture
- difficult to isolate homogenous populations
17
Q
When were adult stem cells discovered?
A
- 1950s
- bone marrow: two different stem cell populations
- haematopoietic stem cells → red and white blood cells, platelets
- bone marrow stromal cells → bone, cartilage, fat, stroma
21
Q
What is reprogramming?
A
- reversing the differentiation process
22
Q
What are methods of reprogramming?
A
- somatic cell nuclear transfer (SCNT)
- SCNT using an embryo at mitosis
- altered nuclear transfer (ANT)
- fusion of skin cells with hESCs
- induced pluripotent stem cells, or iPSCs
23
Q
What is somatic cell nuclear transfer?
A
- first attempts at nuclear reprogramming were in frog eggs, later followed by attempts in mammals
- early studies were followed by several successful cloning experiments using enucleated oocytes and donor nuclei (even ICM nuclei) in a number of livestock species
- key conclusions from successful experiments were:
- egg cytoplasm, but not zygotic cytoplasm was permissive to reprogramming
- eggs must be enucleated to maintain normal ploidy in the developing embryos
- clones have been generated from various foetal and adult cell types, with varying degrees of success
- cloned embryos ≠ fertilised embryos
- poor blastocyst rates
- most cloned embryos die during gestation
- developmental defects
- genome wide gene expression abnormalities
- epigenetic inheritance likely the principle barrier
*
24
Q
What is altered nuclear transfer (ANT)?
A
- eliminate the capacity for a cloned blastocyst to implant normally
- experiments confirmed that this approach is feasible in mouse
- don’t yet know whether the human Cdx2 gene has a similar function in trophoblast development
- scientifically: negates any relationship between the ICM and TE
25
Q
What are iPSCs?
A
- 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
26
Q
How does iPS cell reprogramming occur?
A
- virion
- fusion
- uncoating and reverse transcription
- incorporation of cellular proteins
- intracellular trafficking
- nuclear entry
- access to chromatin
- integration
27
Q
ESC factors responsible for reprogramming, but which ones?
A
- 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
28
Q
What is the contribution of iPSCs?
A
- iPS contribute to all germ layers in vitro and express ESC markers
- but retain somatic memory
29
Q
Have human iPSCs been created?
A
- yes
- induction of pluripotent stem cells from adult human fibroblasts by defined factors
30
Q
What are steps involved in reprogramming?
A
- 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
35
Q
What are disease-specific induced pluripotent stem cells?
A
- iPS Cells derived from somatic cells of patients with genetic disease
36
Q
How could iPSCs be used to treat disease e.g. sickle cell anaemia?
A
- 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
37
Q
What is the process of testing human ES cells for therapy?
A
- 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
