lecture 29: stem and iPS cells

Question 1

What are the different types of stem cells?

Answer 1

• 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

Question 2

What defines a stem cell?

Answer 2

• 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

Question 3

What are differing potencies of stem cells?

Answer 3

• 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

Question 4

What is embryo development?

Answer 4

• 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

Question 5

What are the varying tissue lineages over the course of embryo development?

Answer 5

• fertilised egg ( day 0)
• blastocyst (5)
  
  • trophoblast (6-7)
    
    • cytotrophoblast (8-9)
      
      • syncytioptrophoblast (12)
  • inner cell mass (6-7)
    
    • hypoblast (8-9)
      
      • extraembryonic endoderm (12)
        
        • yolk sac (14)
    • epiblast (8-9)
      
      • amniotic ectoderm (12)
      • primitive ectoderm (12)
        
        • embryonic ectoderm (15)
        • primitive streak (14)
          
          • embryonic endoderm (15)
          • embryonic mesoderm (15)
          • extraembryonic mesoderm (15)

Question 6

What is the history of embryonic stem cells?

Answer 6

• 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

Question 7

What are embryonic stem cells?

Answer 7

• 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

Question 8

What controls self-renewal?

Answer 8

• sox 2
• oct 4
• nanog
• klf 4
• myc

Question 9

What is a model core ES cell regulatory circuitry?

Answer 9

Question 10

What characterises embryonic stem cells?

Answer 10

• morphology
• transcription factor expression
• dependence on glycolytic metabolism and glutaminolysis
• long telomeres, high telomerase activity

Question 11

What characterises pluripotency?

Answer 11

• chimera formation
• differentiation (in vitro, teratoma formation in vivo)

Question 12

What is developmental potential/differentiation?

Answer 12

• 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

Question 13

What is the relationship between developmental potential and epigenetic status?

Answer 13

• 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

Question 14

What are epigenetic mechanisms?

Answer 14

• 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

Question 15

How does the epigenetic response to extrinsic signals occur?

Answer 15

• 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

Question 16

What are adult stem cells?

Answer 16

• 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

Question 17

When were adult stem cells discovered?

Answer 17

• 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

Question 18

How are adult stem cells isolated?

Answer 18

• haematopoietic stem cells
  
  • bone marrow and its peripheral blood
  • placenta and umbilical cord
• mesenchymal stem cells
  
  • bone marrow of the iliac crest or femoral head
  • adipose tissue
• identified by surface marker expression

Question 19

Where have ASCs been found?

Answer 19

• many different tissues: blood/bone marrow, heart, fat, epidermis, retina, dental pulp
• bone marrow stem cells (MSCs)
  
  • widely used for transplantation (HLA compatibility)
• adipose tissue stem cells
  
  • differentiated towards functional cardiomyocytes, osteoblasts, haematopoietic and neural cells
• cord blood stem cells
  
  • transplantation is an accepted curative therapy and non-malignant inherited diseases
  • useful for child transplantations, hampered in adults by low cell dose
• disadvantages:
  
  • cells move away from the transplantation site
  • cell integration is not significant/cell death

Question 21

What is reprogramming?

Answer 21

• reversing the differentiation process

Question 22

What are methods of reprogramming?

Answer 22

• 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

Question 23

What is somatic cell nuclear transfer?

Answer 23

• 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
    *

Question 24

What is altered nuclear transfer (ANT)?

Answer 24

• 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

Question 25

What are iPSCs?

Answer 25

• 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

Question 26

How does iPS cell reprogramming occur?

Answer 26

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

Question 27

ESC factors responsible for reprogramming, but which ones?

Answer 27

• 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

Question 28

What is the contribution of iPSCs?

Answer 28

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

Question 29

Have human iPSCs been created?

Answer 29

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

Question 30

What are steps involved in reprogramming?

Answer 30

• 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

Question 31

What is the difference between ESCs and iPSCs in mouse?

Answer 31

• 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

Question 32

What is the difference between iPSCs and ESCs in human?

Answer 32

• 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

Question 33

A new route to human embryonic stem cells: a game changer?

Answer 33

• 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)

Question 34

What is the “best” source of pluripotent cells for therapeutics?

Answer 34

• 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

Question 35

What are disease-specific induced pluripotent stem cells?

Answer 35

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

Question 36

How could iPSCs be used to treat disease e.g. sickle cell anaemia?

Answer 36

• 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

Question 37

What is the process of testing human ES cells for therapy?

Answer 37

• 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

Question 38

What are unregulated stem cell based treatments?

Answer 38

• 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

Question 39

What is an example of a succesful stem cell therapy?

Answer 39

• first fully synthetic organ transplant saves cancer patient

Question 40

What are important considerations of stem cell therapies?

Answer 40

• 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

Question 41

summary

Answer 41

• 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