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Genetic Engineering > Test 2 > Flashcards

Test 2 Flashcards

1
Q

What tissues have high regenerative capabilities in humans? Moderate? Low?

A

High: skin, intestines, liver
Mod: bone, muscle
Low: heart, connective tissue, brain

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2
Q

Basic characteristics of stem cells

A

Self-renewal

Differentiation/multi-potency

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3
Q

Embryonic Stemcells are:

a) Totipotent
b) Pluripotent
c) Unipotent

A

Pluripotent

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4
Q

Totipotent

A

Can for any tissue, including placenta

Cannot reproduce by themselves

Therefore, cannot be stem cells

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5
Q

Pluripotent

A

Can form any tissue in the embryo, but not the placenta

Embryonic stem cells from blastocyst (or induced pluripotent)

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6
Q

Multipotent

A

Can form multiple cell types within a particular issue, organ, or physiological system.

(Somatic or adult stem cells)

Self-renewal is limited

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7
Q

Cell differentiation

A

Unspecialized cells develop into specialized cells

Same genetic material, but different genes are turned on in different cells

Different cell lineages determined by enviroment

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8
Q

Commitment

A

Epigenetic modification = genetic memory

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9
Q

Epigenetic components

A

1) DNA methylation
2) Histone modification

If goes wrong may form teratoma

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10
Q

DNA methylation

A

DNA methyltransferases methylate CPG islands

Silenced (condensed) when methylated

Transcription occurs when unmethylated oracetylated

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11
Q

Character of adult stem cells: primary role

A

Maintain and repair the tissue in which they are found

Produce limited kinds of cells

Difficult to identity amoung other cells and population is small in tissue

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12
Q

Character of adult stem cells: decreased self renewal/proliferation

A

Telomeres are shorter than in embryonic stem cells

No telomerase = senescence

(plasmids are circular to prevent degradation, no site for exonuclease cutting like Dnase I or II)

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13
Q

Kinds of adult stem cells

A 
Hematopoietic stem cells
Mesenchymal stem cells
Neural stem cells
Epithelial stem cells
(digestive lining)
(epidermis)
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14
Q

Mesenchymal stem cells: characteristics

A

Easy isolation, high expansion, reproducible (advantage)

Source: bone marrow, adipose tissue,
peripheral blood, umbilical cord

Differentiation pathway: Osteogenic, chondrogenic, adipogenic

Culture condition:
Tissue culture treated surface with
special FBS

Differentiation:FGF, RGF,TGF-beta,
BMPs

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15
Q

Mesenchymal stem cell: lineage

A

Already commited: will develop bone or other mesenchyme tissue wherever injected

Bone, Cartilage, Muscle, Marrow, Tendon/Ligament, Connective tissue

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16
Q

Neural stem cells (NSCs): source

A

SVZ
(from the olfactory bulb and migrate)
(when tissue damage, stem cells migrate to injury and lose sense of smell)

Granule layer of dentate gyrus in hippocampus (50 1st dates)

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17
Q

Neural stem cells (NSCs): differentiation lineages

A

Neurons, astorcytes, oligodendrocytes

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18
Q

Neural stem cells (NSCs): maintenance cell culture

A

EGF, FGF without serum to form neurospheres

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19
Q

Neural stem cells (NSCs): differentiation cell culture

A

Serum, NGF, BDNF

Unknown factors in serum impt, but not defined

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20
Q

Adult neurogenesis: HIppocamus

A

Dentate gyrus in hippocampus regenerates easily due to stem cells present

  1. Proliferation
  2. Fate determination
  3. Migration
  4. Integration
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21
Q

Neural Stem Cells (NSCs): migration

A

Normal activity:
From ventricular and SVZ (in the wall of the lateral ventricle adjacent to the cautate-putamen) to olfactory bulb and differentiation into local interneurons

From sub-granular zone of the hippocampus (but also in the cerebral cortex and SZ) to dentate gyrus

In many adult tissues, cell loss from natural attrition or injury is balanced by proliferation and subsequent differentiation of multipotential germinal cells termed stem cells.

Evidence suggests that the brain, like many other tissues, is in a state of dynamic equillibrium. It has an endogenous population of stem cells that proliferate in response to environmental and pharmacological manipulations and that can replace cells lost in some experimental lesions.

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22
Q

Adult neurogensis: SVZ/OB system

A

1) Proliferation/fate determination (SVZ)
2) Migration (RMP)
3) Integration (OSN - olfactory)

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23
Q

Migration for repair from endogenous stem cells

A

Somal translocation

Can see bipolar morphology

Increased number of neural stem cells after stroke - because stem cells from olfactory bulb migrate to repair injury

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24
Q

Neural Stem Cells (NSCs): differentiation lineage pathway

A

Stem cell –>
Progenitor –>
etc

Neurons (projecting, inter-)
Astrocytes
Ogliodendrocytes
------------------------------------
Microglia (immunological)
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25
Q

Disadvantages to 1* cells

A

Immune respones

Live cell donors

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26
Q

Regeneraton in Nature

A

Outstanding Examples:
Planarian
Crayfish
Embryos

Inverse Relationship:
Increase complexity = decrease regenerative ability

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27
Q

Hematopoietic Stem Cells (HSCs)

A

Source: bone marrow , peripheral blood

Differentiation pathways: Blood cells including T cells, B cells and Erythrocyte

Culture conditions:
Maintenance: culture with activation of cytokine receptors by IL-3, IL-6
Differentiation: granulocyte-colony
stimulating factor (G-CSF), erythropoietin (EPO)

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28
Q

Transplantation of hNSCs in APP23 mice: spatial memory experiment

A

Environmental ques to memorize location of platform

Aged mice have slight decreased spatial memory over time when compare to young

Alz-model mice have significantly decreased spatial memory
(hNSCs transplanted into SVZ)

Increase in spatial memory, even surpassing the young animal

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29
Q

List any 3 Adult Stem Cells used to treat heart diseases.

A

From heart: Sca-1 (+) cells, c-kit (+) cells, SP (special population) cells

From bone marrow: human mesenchymal stem cells

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30
Q

List 2 methods of delivering adult stem cells into the heart.

A

Direct: transcoronary sinus, transendocardial, intramyocardial, epicardial collagen/stem cell patch

Indirect: intravenous, intracoronary w/balloon catheter, tail vein

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31
Q

List 4 cell types derived from Mesenchymal Stem Cells.

A 
Chondrocytes (cartilage)
Myocytes (muscles)
Fibroblasts (skin, tendon, ligaments)
Osteocytes (bone)
Adipocytes (fat)
Stromal cells (marrow) 
Astrocytes (CNS)
32
Q

Describe 2 delivery methods for bone regeneration.

A

Fracture site delivery:
MSCs are placed in a bio-engeenered scaffold that mimics bone structure and directly injected in the fracture site along with bone differentiation factors such as: BMPs and PRP.

Systemic delivery :
MSCs are delivered into circulation through intravenous injection with or without bone differentiation factors such as PTH, Sclerotin Ab, DKK1 Ab and IGF.

33
Q

Describe phase 1, 2 & 3 skin burn disease therapy in humans.

A

Phase I: Utilizing a mixed skin cell preparation, including the patient’s skin stem cells, intra-operative isolation and direct application

Phase II: Cell application with skin cell spray gun

Phase III: Cell and wound support with temporary artificial wound capillary system under the wound dressing.

Artifical skin can also be made using stem cells from umbilical cord, fibrin, and agarose.

34
Q

What are pluripotent stem (iPS) cells?

A

Pluripotent embryonic stem cell-like cells

Generated with different transcription factors

Reprogramming somatic cells (eg. skin fibroblast)

Embryonic stem (ES) cells are immortal, pluripotent cells

Derived from inner cell mass (ICM) of the pre-implantation blastocyst

35
Q

What are different stem cells?

A

Embryonic Stem Cells: from embryos created by fertilization or by cloning (somatic cell nuclear transfer)

Induced Pluripotent stem cells: from normal cells that are reprogrammed to behave like embryonic stem cells

Adult Stem Cells: stem cells normally found in body tissues from birth onward, as well as teh unbilical cord, etc.

36
Q

Commonly used transcription factors

A

Oct3/4, Sox2, c-Myc, Klf4, Nanog, Lin28

37
Q

Source of cells to induce iPS cells

A

Skin fibroblasts
Hepatocytes
Blood cells
Cardiomyoblasts

38
Q

Pluripotency Determination

A

Form embyroid bodies in vitro

Form teratomas in vivo

39
Q

Can iPS cells differentiate into the 3 germ layers? What are they?

A

Yes.

Mesoderm, endoderm, ectoderm

40
Q

iPS cell generation

A
  1. Somatic cells (eg. skin fibroblasts) removed from host
  2. Plated on tissue culture plate
  3. Cells exposed to transcriptional factors (viral or non-viral methods)
  4. Cells moved to place with MEFs to keep in pluripotent/self-renewal stage
  5. Embryonic stem cell-like colony formation
41
Q

What are classical methods of reprogramming cells? How do new methods differ?

A

Classical method: Oct4/Sox2/Klf4/c-Myc delivered via retrovirus

New methods: fewer factors, chemicals, non-integrated vectors

42
Q

What are the characteristics of fully v. partially v. aberrantly reprogrammed cells?

A

Fully reprogrammed iPS cells: germline-competent (mouse), teratoma-competent (human, mouse)

Partially reprogrammed iPS cells:
self-renew, in vitro differentiation

Aberrantly reprogrammed cells: self-renew, refractory to differentiation

43
Q

Describe the method of generating iPS cells: viral strategies

A
  1. Cell isolation/cultivation
  2. Transfection with factors (+/-epigenetic modification by small molecules)
  3. Selection of iPS colonies
  4. Propagation of iPS colonies (+/- removal of viral vectors)
44
Q

What are the applications of iPS cells?

A

Cell transplantation

Toxicity testing

Disease models

Characterization:
Epigenetic analysis
Gene-specific analysis
Chromosome- and genome-wide analysis

45
Q

Viral v. non-viral methods

A

Sendai virus:
Increased reprogramming efficiency for even more colonies

Lower cytoxicity to allow for smaller starting cell population

Faster clearance to get to your iPS cell experiments sooner

46
Q

What are 3 viruses used for transvection of reprogramming plasmids?

A

Retrovirus
Lentivirus
Sendai
Nonintegrating virus

47
Q

What are nonviral methods of reprogramming somatic cells?

A

Plasma vector based
Chemicals
Small molecules
miRNA

48
Q

Describe the plasmid vector-based method of reprogramming.

A

Plasmids are DNA molecules that replicate independently of, the chromosomal DNA.

Designed to stay separate from the host cell’s genome.

2A self-cleavage sequences express Oct3/4, Sox2, and Klf4 in a single expression vector.

49
Q

Describe the the microRNA method of reprogramming.

A

Small noncoding RNAs

Critical for the expression control of more than a third of all protein coding genes

Bind to the 3 untranslated region (UTR) of target mRNAs via an imperfect match to repress their translation and/or stability

50
Q

Issues with viral methods of reprogramming

A

Viruses can integrate into the genome of non-dividing cells.

Inserting virus DNA that combines with a cell’s DNA can trigger that cell to become cancerous.

Although integrated viruses transcriptionally silenced following reprogramming, re-expression reprogramming factors may interfere with differentiation and subsequent cell behavior.

51
Q

Issues with non-viral methods of reprogramming

A

Inefficiency
Costly
Time consuming

52
Q

How do you confirm pluripotency?

A

Morphology

Transcription factor expression (Western blot and ICC)

Alkaline phosphotase staining (doesn’t stain differentiated cells)

53
Q

How do iPS cells differentiate?

A

In vitro differentiation:
Cell culture –> specific markers –> functional properties

In vivo chimeric tissue:
Teratoma formation –> chimerism –> germ line

In situ regenerative potental:
Lineage specification –> delivery –> engraftment –> functional recovery

54
Q

iPS cell derived cell types

A 
Cardiomyoctes
Adipocytes
Neural cells (dopamenerigc neurons, motor neurons)
Pancreatic B-cells
Hematopoietic Progenitor Cells
55
Q

Similarities between iPS and ES cells

A 
Pluripotent
Can generate all three germ layers
Morphology
Gene expression
Teratoma formation
56
Q

Healthy donor v. Donor with disease/Generation of patient specific cells

A
  1. Harvest human somatic cells:
  2. Reprogramming
  3. Selection/progation
  4. iPS cells

Heathy cells –> iPS cells
Disease specific cells –> disease iPS cells

57
Q

Application Potential of iPS cells

A 
Regenerative medicine
Cell replacement therapy
Human pathology modeling (pathogensis, disease-associated genes)
Drug target discovery/screening
Toxicity/toxicology testing
Cell differentiation
58
Q

Disease modeling

A

Cells can be obtained from a patient with almost any disease, reprogrammed to a pluripotent state, expanded in vitro, and differentiated into a cell type that expresses features of the disease. Such disease models “in a dish” can be used to test new drugs and gene therapies.

Generation of disease-specific pluripotent cells capable of differentiation into the various tissues affected in each condition.
Provides new insights into disease pathophysiology by permitting analysis in a human system, under controlled conditions in vitro.

59
Q

Describe the steps necessary to model a human heart disorder by generating patient-specific iPS cell lines for studying the disease phenotype

A

Control + Patient

  1. Harvest somatic cells
  2. Reprogram into iPS cells
  3. Differentiate into cardiomyocytes
  4. Phenotypic analysis (eg. action potential recording, calcium imaging)
60
Q

Cardiomyocyte transplant

A

Reversal of complications from myocardial infarction: regenerate myocardial tissue, inhibit apoptosis, and improve cardiac function in the infarcted hearts

61
Q

B-cell transplant

A

iPS cell-derived B-cell-filled immunoprotective device transplanted into patient will secrete insulin relieving them of the need for insulin injections returning them to “normal”.

Also can be used for drug screening.

Reversal of hyperglycemia in type-1 and type-2 diabetic mouse models.

Possible treatment for type 1 and type 2 diabetes.

62
Q

Generation of liver disease-specfic iPS cells

A
  1. Skin/liver/blood harvest
  2. Reprogramming
  3. Patient-specific iPS cells
  4. Disease modeling or gene correction

Disease modeling:

a. Differentiate into hepatocytes
b. Drug screening/pathogenesis
c. Disease and patient-specific drugs

Gene correction:

a. Repaired iPS cells
b. Differentiate into hepatocytes
c. Transplantation into patient

63
Q

Genetically corrected iPS cells to cure sickle cell anemia

A

Reprogrammed, repaired iPS cells are differentiated into blood cells and transplanted into diseased mouse

64
Q

Advantages and Challenges of iPS cell-derived transplants

A

Removes ethical issues

Removes issues with immune responses

Like ES cells, teratoma formation still an issue

Inefficiency of the reprogramming process

Directing differentiation to achieve specific cell type

Reliance on viral vectors

65
Q

Adult stem cell applications

A

Brain: stroke, traumatic brain injury, learning defects, Alzheimer’s diseases, Parkinson’s disease

Missing teeth
Amytrophic lateral sclerosis (ALS)
Muscular dystrophy
Cirrhosis
Kidney cancer
Osteoarthritis rheumatoid arthritis
Spinal cord injury
Baldness 
Blindness
Deafness
Lung cancer
Acute heart damage
Genetic bone disease
Diabetes type 2
Crohn's disease
Prostate disease
66
Q

List 3 challenges to stem cell therapies in cardiac disease.

A

Isolation:
Purity of isolated cells
Sufficient number of cells
Differentiation into cardiomyoctyes before transplantation

Delivery:
Safety
Cell retention
Spatial distribution

Survival and proliferation:
Ischaemic environment
Inflammation
Immune response
Fibrosis
Growth and adhesion signals
Formation of functional blood vessels

Electromechanical integration:
Differentiation into mature cardiomyocytes
Electrical integration
Mechanical coupling

Stability and safety:
Long-term engraftment
Arrythmogenicity

67
Q

Regeneration of MI heart with BMCs

A

BMCs = c-kit (+) and carry Y-chromosome (to identify in female mice), express Nkx2.5 and Cnx-43

BMCs acquire the cardiogenic fate by forming heart cells (cardiac myoctes, vascular smooth cells, and endothelial cells)

68
Q

Fabrication of Bone from MSCs

A
  1. Proliferation
  2. Osteoblasts + porous ceramics => bone matrix formation, chondrocytes + polymer => cartlagenous matrix formation
  3. Cell differentiation
  4. Fabricaiton of bone/cartilage composites
69
Q

Direct effect of bone regeneration using MSCs

A

MSCs engraft in fracture site and differentiate into major bone cells: Osteoblasts and Chondrocytes

70
Q

Indirect effect of bone regeneration using MSCs

A
  1. MSCs secrete cytokines that promote bone repair.
  2. Induce angiogenesis.
  3. Regenerate extracellular proteins and factors. Produce matrix.
  4. Inhibit inflammation at site of injury.
  5. MSCs can be used to deliver genes to the site of injury to promote healing.
71
Q

MSCs to repair broken bone

A
  1. Segmental bone defect occurs
  2. A collagen scaffold is implanted into the defect site
  3. Endogenous MSCs invade and implanted scaffold
  4. A BMP gene is injected into the MSC-populated scaffold
  5. An ultrasound pulse is applied to the defect site leading to DNA uptake by MSCs. BMP secretion leads to fracture repair
72
Q

Umbelical cord-MSCs for the treatment of skin burns

A

GFP labeled hUC-MSCs or PBS was intravenous injected into respective groups.

Burn transplanted hUC-MSCs healed better than burn group injected with PBS. ( :( )

73
Q

Reprogramming of somatic cells to a pluripotent state: reprogramming process

A

Somatic cell + Oct4/Sox2/c-Myc/Klf4

Sequence of stochastic epigenetic events:

  1. Partially reprogrammed - retroviruses expressed, endogenous Oct4 locus not reprogrammed
  2. Fully reprogrammed - Dnmt3a, b activated, retroviruses silenced, endogenous Oct4/Nanog loci reprogrammed
74
Q

Factors to direct differentiation of SiPS cells: dopaminergic, cholinergic, inner hair

A

Dopaminergic: Nurr1
Cholinergic: Lxh8
Inner hair: Hath1/Atoh1

75
Q

Long-term: ‘clinical-grade’ cells

A

No contamination: animal-substance free conditions
- GMP, human feeder cells, autologous serum, feeder-free culture, chemically -defined media

No immune rejection: no immunosupressive drugs
- use of autologous cells, somatic cell nuclear transfer, genetic engineering, reprogramming

Functionality: production of cells with target functions

  • understand effects of normal/disease environment of the cells
  • create optimal environment, tissue engineering approach
76
Q

Cell separation technologies

A

Isopyncnic entrifugation: density

Filtration: size

Flow Cytometry (FACS): size, granularity, fluorescence

Batch affinity systems: antibody, avidin-biotin

Batch magnetic systems: paramagnetic difference

Genetic Engineering (4 decks)

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