lecture 29: stem and iPS cells Flashcards Preview

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Flashcards in lecture 29: stem and iPS cells Deck (41):
1

What are the different types of stem cells?

  • 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

What defines a stem cell?

  • 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

What are differing potencies of stem cells?

  • 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

What is embryo development?

  • 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

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

  • 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)

6

What is the history of embryonic stem cells?

  • 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

What are embryonic stem cells?

  • 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

What controls self-renewal?

  • sox 2 
  • oct 4 
  • nanog 
  • klf 4 
  • myc 

9

What is a model core ES cell regulatory circuitry?

10

What characterises embryonic stem cells?

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

11

What characterises pluripotency?

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

12

What is developmental potential/differentiation?

  • 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

What is the relationship between developmental potential and epigenetic status?

  • 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

What are epigenetic mechanisms?

  • 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

How does the epigenetic response to extrinsic signals occur?

  • 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

What are adult stem cells?

  • 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

When were adult stem cells discovered?

  • 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 

18

How are adult stem cells isolated?

  • 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 

19

Where have ASCs been found?

  • 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 

20

21

What is reprogramming?

  • reversing the differentiation process 

22

What are methods of reprogramming?

  • 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

What is somatic cell nuclear transfer?

  • 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

What is altered nuclear transfer (ANT)?

  • 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

What are iPSCs?

  • 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

How does iPS cell reprogramming occur?

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

27

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 
  • 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

What is the contribution of iPSCs?

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

29

Have human iPSCs been created?

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

30

What are steps involved in reprogramming?

  • 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 

31

What is the difference between ESCs and iPSCs in mouse?

  • 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 

32

What is the difference between iPSCs and ESCs in human?

  • 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 

33

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

  • 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) 

34

What is the "best" source of pluripotent cells for therapeutics?

  • 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 

35

What are disease-specific induced pluripotent stem cells?

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

36

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

  • 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

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

  • 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 

38

What are unregulated stem cell based treatments?

  • 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

39

What is an example of a succesful stem cell therapy?

  • first fully synthetic organ transplant saves cancer patient 

40

What are important considerations of stem cell therapies?

  • 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 

41

summary

  • 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