Test 2 Flashcards
What tissues have high regenerative capabilities in humans? Moderate? Low?
High: skin, intestines, liver
Mod: bone, muscle
Low: heart, connective tissue, brain
Basic characteristics of stem cells
Self-renewal
Differentiation/multi-potency
Embryonic Stemcells are:
a) Totipotent
b) Pluripotent
c) Unipotent
Pluripotent
Totipotent
Can for any tissue, including placenta
Cannot reproduce by themselves
Therefore, cannot be stem cells
Pluripotent
Can form any tissue in the embryo, but not the placenta
Embryonic stem cells from blastocyst (or induced pluripotent)
Multipotent
Can form multiple cell types within a particular issue, organ, or physiological system.
(Somatic or adult stem cells)
Self-renewal is limited
Cell differentiation
Unspecialized cells develop into specialized cells
Same genetic material, but different genes are turned on in different cells
Different cell lineages determined by enviroment
Commitment
Epigenetic modification = genetic memory
Epigenetic components
1) DNA methylation
2) Histone modification
If goes wrong may form teratoma
DNA methylation
DNA methyltransferases methylate CPG islands
Silenced (condensed) when methylated
Transcription occurs when unmethylated oracetylated
Character of adult stem cells: primary role
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
Character of adult stem cells: decreased self renewal/proliferation
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)
Kinds of adult stem cells
Hematopoietic stem cells Mesenchymal stem cells Neural stem cells Epithelial stem cells (digestive lining) (epidermis)
Mesenchymal stem cells: characteristics
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
Mesenchymal stem cell: lineage
Already commited: will develop bone or other mesenchyme tissue wherever injected
Bone, Cartilage, Muscle, Marrow, Tendon/Ligament, Connective tissue
Neural stem cells (NSCs): source
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)
Neural stem cells (NSCs): differentiation lineages
Neurons, astorcytes, oligodendrocytes
Neural stem cells (NSCs): maintenance cell culture
EGF, FGF without serum to form neurospheres
Neural stem cells (NSCs): differentiation cell culture
Serum, NGF, BDNF
Unknown factors in serum impt, but not defined
Adult neurogenesis: HIppocamus
Dentate gyrus in hippocampus regenerates easily due to stem cells present
- Proliferation
- Fate determination
- Migration
- Integration
Neural Stem Cells (NSCs): migration
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.
Adult neurogensis: SVZ/OB system
1) Proliferation/fate determination (SVZ)
2) Migration (RMP)
3) Integration (OSN - olfactory)
Migration for repair from endogenous stem cells
Somal translocation
Can see bipolar morphology
Increased number of neural stem cells after stroke - because stem cells from olfactory bulb migrate to repair injury
Neural Stem Cells (NSCs): differentiation lineage pathway
Stem cell –>
Progenitor –>
etc
Neurons (projecting, inter-) Astrocytes Ogliodendrocytes ------------------------------------ Microglia (immunological)
Disadvantages to 1* cells
Immune respones
Live cell donors
Regeneraton in Nature
Outstanding Examples:
Planarian
Crayfish
Embryos
Inverse Relationship:
Increase complexity = decrease regenerative ability
Hematopoietic Stem Cells (HSCs)
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)
Transplantation of hNSCs in APP23 mice: spatial memory experiment
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
List any 3 Adult Stem Cells used to treat heart diseases.
From heart: Sca-1 (+) cells, c-kit (+) cells, SP (special population) cells
From bone marrow: human mesenchymal stem cells
List 2 methods of delivering adult stem cells into the heart.
Direct: transcoronary sinus, transendocardial, intramyocardial, epicardial collagen/stem cell patch
Indirect: intravenous, intracoronary w/balloon catheter, tail vein
List 4 cell types derived from Mesenchymal Stem Cells.
Chondrocytes (cartilage) Myocytes (muscles) Fibroblasts (skin, tendon, ligaments) Osteocytes (bone) Adipocytes (fat) Stromal cells (marrow) Astrocytes (CNS)
Describe 2 delivery methods for bone regeneration.
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.
Describe phase 1, 2 & 3 skin burn disease therapy in humans.
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.
What are pluripotent stem (iPS) cells?
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
What are different stem cells?
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.
Commonly used transcription factors
Oct3/4, Sox2, c-Myc, Klf4, Nanog, Lin28
Source of cells to induce iPS cells
Skin fibroblasts
Hepatocytes
Blood cells
Cardiomyoblasts
Pluripotency Determination
Form embyroid bodies in vitro
Form teratomas in vivo
Can iPS cells differentiate into the 3 germ layers? What are they?
Yes.
Mesoderm, endoderm, ectoderm
iPS cell generation
- Somatic cells (eg. skin fibroblasts) removed from host
- Plated on tissue culture plate
- Cells exposed to transcriptional factors (viral or non-viral methods)
- Cells moved to place with MEFs to keep in pluripotent/self-renewal stage
- Embryonic stem cell-like colony formation
What are classical methods of reprogramming cells? How do new methods differ?
Classical method: Oct4/Sox2/Klf4/c-Myc delivered via retrovirus
New methods: fewer factors, chemicals, non-integrated vectors
What are the characteristics of fully v. partially v. aberrantly reprogrammed cells?
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
Describe the method of generating iPS cells: viral strategies
- Cell isolation/cultivation
- Transfection with factors (+/-epigenetic modification by small molecules)
- Selection of iPS colonies
- Propagation of iPS colonies (+/- removal of viral vectors)
What are the applications of iPS cells?
Cell transplantation
Toxicity testing
Disease models
Characterization:
Epigenetic analysis
Gene-specific analysis
Chromosome- and genome-wide analysis
Viral v. non-viral methods
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
What are 3 viruses used for transvection of reprogramming plasmids?
Retrovirus
Lentivirus
Sendai
Nonintegrating virus
What are nonviral methods of reprogramming somatic cells?
Plasma vector based
Chemicals
Small molecules
miRNA
Describe the plasmid vector-based method of reprogramming.
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.
Describe the the microRNA method of reprogramming.
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
Issues with viral methods of reprogramming
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.
Issues with non-viral methods of reprogramming
Inefficiency
Costly
Time consuming
How do you confirm pluripotency?
Morphology
Transcription factor expression (Western blot and ICC)
Alkaline phosphotase staining (doesn’t stain differentiated cells)
How do iPS cells differentiate?
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
iPS cell derived cell types
Cardiomyoctes Adipocytes Neural cells (dopamenerigc neurons, motor neurons) Pancreatic B-cells Hematopoietic Progenitor Cells
Similarities between iPS and ES cells
Pluripotent Can generate all three germ layers Morphology Gene expression Teratoma formation
Healthy donor v. Donor with disease/Generation of patient specific cells
- Harvest human somatic cells:
- Reprogramming
- Selection/progation
- iPS cells
Heathy cells –> iPS cells
Disease specific cells –> disease iPS cells
Application Potential of iPS cells
Regenerative medicine Cell replacement therapy Human pathology modeling (pathogensis, disease-associated genes) Drug target discovery/screening Toxicity/toxicology testing Cell differentiation
Disease modeling
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.
Describe the steps necessary to model a human heart disorder by generating patient-specific iPS cell lines for studying the disease phenotype
Control + Patient
- Harvest somatic cells
- Reprogram into iPS cells
- Differentiate into cardiomyocytes
- Phenotypic analysis (eg. action potential recording, calcium imaging)
Cardiomyocyte transplant
Reversal of complications from myocardial infarction: regenerate myocardial tissue, inhibit apoptosis, and improve cardiac function in the infarcted hearts
B-cell transplant
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.
Generation of liver disease-specfic iPS cells
- Skin/liver/blood harvest
- Reprogramming
- Patient-specific iPS cells
- 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
Genetically corrected iPS cells to cure sickle cell anemia
Reprogrammed, repaired iPS cells are differentiated into blood cells and transplanted into diseased mouse
Advantages and Challenges of iPS cell-derived transplants
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
Adult stem cell applications
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
List 3 challenges to stem cell therapies in cardiac disease.
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
Regeneration of MI heart with BMCs
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)
Fabrication of Bone from MSCs
- Proliferation
- Osteoblasts + porous ceramics => bone matrix formation, chondrocytes + polymer => cartlagenous matrix formation
- Cell differentiation
- Fabricaiton of bone/cartilage composites
Direct effect of bone regeneration using MSCs
MSCs engraft in fracture site and differentiate into major bone cells: Osteoblasts and Chondrocytes
Indirect effect of bone regeneration using MSCs
- MSCs secrete cytokines that promote bone repair.
- Induce angiogenesis.
- Regenerate extracellular proteins and factors. Produce matrix.
- Inhibit inflammation at site of injury.
- MSCs can be used to deliver genes to the site of injury to promote healing.
MSCs to repair broken bone
- Segmental bone defect occurs
- A collagen scaffold is implanted into the defect site
- Endogenous MSCs invade and implanted scaffold
- A BMP gene is injected into the MSC-populated scaffold
- An ultrasound pulse is applied to the defect site leading to DNA uptake by MSCs. BMP secretion leads to fracture repair
Umbelical cord-MSCs for the treatment of skin burns
GFP labeled hUC-MSCs or PBS was intravenous injected into respective groups.
Burn transplanted hUC-MSCs healed better than burn group injected with PBS. ( :( )
Reprogramming of somatic cells to a pluripotent state: reprogramming process
Somatic cell + Oct4/Sox2/c-Myc/Klf4
Sequence of stochastic epigenetic events:
- Partially reprogrammed - retroviruses expressed, endogenous Oct4 locus not reprogrammed
- Fully reprogrammed - Dnmt3a, b activated, retroviruses silenced, endogenous Oct4/Nanog loci reprogrammed
Factors to direct differentiation of SiPS cells: dopaminergic, cholinergic, inner hair
Dopaminergic: Nurr1
Cholinergic: Lxh8
Inner hair: Hath1/Atoh1
Long-term: ‘clinical-grade’ cells
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
Cell separation technologies
Isopyncnic entrifugation: density
Filtration: size
Flow Cytometry (FACS): size, granularity, fluorescence
Batch affinity systems: antibody, avidin-biotin
Batch magnetic systems: paramagnetic difference
