- Mix of stem & progenitor cells (totipotent/pluripotent) - Give rise to limbs by redifferentiation

Exam #3 Flashcards by Nicole Arbelaez

Identify/choose the most common proteins involved in regeneration from the list

a. Hox Genes
b. BMP (all)
c. Wnt
d. APC (adenomatous polyposis coli – signaling mechanism)

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Define Blastema

Mix of stem & progenitor cells (totipotent/pluripotent)
Give rise to limbs by redifferentiation

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Define Transdifferentiation

Differentiation between differentiated cells

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Match tissue & their protein signals

Limb (FGF8 - anterior & Shh - posterior)
Iris (FGF2 & BMP must be inhibited)

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Name any 4 causes of liver failure

Genetics, Acetaminophen overdose, Alcohol, Obesity

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Name any 4 cell types found in the liver

a. Hepatocytes – specialized epithelial cell that makes up most of liver
b. Hepatocyte Stellate Cells – progenitor stem cells that respond to injury
c. Hepatic sinusoidal endothelial cells – inflammation regulation, secrete anti-inflammatory molecules
d. Hepatoblasts – progenitor cells (similar to stellate – transdetermination)

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Why are cholangiocytes important in the liver?

Secrete bile and important for homeostasis (change in pH)

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Remember the pro-regenerative signals active during liver damage. (VEGF, HGF, Wnt)

a. VEGF is active during normal division and liver cell maintenance
b. Acts as pro-regenerative signal along with HGF and Wnt2

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Explain the process of clinical trials required for stem cell treatment approval

Safety (animal testing, phase 1)
Efficacy (Phase 2)
Longterm Safety and efficacy (Phase 3)
Safety monitoring (Phase 4)

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Mention any 2 pros and cons of using MSCs, ESCs and iPSCs for clinical applications. Which cell type might be approved 1st and why?

MSC (Adult Stem) – most likely to be approved 1st
Pro – thought to be less likely to be rejected if used for transplants, success has been demonstrated in various applications
Cons – Limitations on ASC ability to differentiate, cant be grown for long periods of time

ESC
Pros – Maintained and grown for 1 year or more, established protocols for maintenance in culture
Cons – Process to generate is inefficient, unsure whether they would be rejected if used in transplants

iPSCS
Pros – abundant somatic cells of donor can be used, issues of histocompatibility can be avoided
Cons – Methods for maintenance of differentiated cells are not certain, viruses are used to introduce embryonic genes and has been shown to cause cancer in mouse

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What are nucleases? Mention the 3 most common nucleases to edit stem cells

Nucleases – enzyme that cleaves a chain of nucleotides in nucleic acids into smaller units

ZFNs – zinc finger nucleases
TALENS – transcription activator-like effector nucleases
CRISPR – clustered regularly interspace short palindrome repeats

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Compare between ZFNs, TALENS and CRISPR. Which is the most efficient enzyme and why? (4 per)

ZFNs (nuclease)
- Fok 1 (endonuclease) to cleave DNA
- Non-specific and cleaves outside recognition sites
- High rates of off-target breaks
- High cytotoxicity & target issue

TALENS (nuclease)
- Fok1 to cleave DNA
- More conserved repeats
- Easy to design and construct
- Lower cytotoxicity

CRISPR (most efficient enzyme)
- Bacterial adaptive immunity
- Cas9 is used
- Higher efficiency
- Easily designed, more accurate

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How does CRISPR work? Explain the function of Cas9, gRNA, PAM

Cas9
- Associated with CRISPR (nuclease)
- Endonuclease (Makes the cut)

gRNA
- Is used to find complementary DNA so that Cas9 can make the cut

PAM
- Protospacer Adjacent Motif
- Sequence 3 nt

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Who won the Nobel prize for the discovery of CRISPR?

Emmanuelle Charpentier and Jennifer Doudna share the award for devolving the precise genome-editing technology

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Briefly explain how CRISPR is used for Knock-in and knock-out models in stem cells (HR and NHEJ)

Knock- in
- Inducing mutations
- Correct mutations

Knock-out
- Inducing mutation

HDR
- Homology Directed Repair
- Gene Repair or mutations
- Precise repair mechanism that uses homologous donor DNA to repair DNA damage

NHEJ
- Non-Homologous End Joining
- Gene disruption
- Error-prone mechanism in which broken ends of DNA are joined together

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Explain the mechanism/selection of CRISPR edited stem cells with diagram.

a. Bacterial Adaptive Immunity
b. Uses s-RNA guide to find complementary DNA sequence to cleave
c. Cas9 endonuclease to make cut
d. PAM (protospacer adjacent motif) sequence (3 nucleotides)

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Describe the fix and swap mechanism of b-Thalassemia using CRISPR. Which one is more clinically translatable and why?

Fix Mechanism
- Using CRISPR and a DNA template to fix the mutation in the hemoglobin gene
- Alpha and beta

Swap Mechanism
- CRISPR reactivate fetal hemoglobin gene by turning off the BCL11A gene (for adult hemoglobin gene)
- Alpha and gamma

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How was the 1st CRISPR baby produced to negate HIV?

Using IVF (HIV+ parents egg+sperm), culture embryo then use CRISPR/Cas9 to knock out CCR5 gene (core receptor that binds w/ HIV for entry into Th cell). Then implantation of the gene-edited embryo to mother/surrogate, HIV free baby is born
HIV Infection in babies: gp120 on HIV cant bind to CCR5 (co-receptor) meaning that although it can bind to CD4 receptor, it needs both to enter cell.

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Any 2 advantages and disadvantages of CRISPR in treating HIV

Advantages
1. HIV protection/prevention
2. Decreased inflammatory responses and fibrosis

Disadvantages
1. Reduced protection against influenza
2. Early manifestation of West Nile Infection

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CAR-T cell therapy is a more precise way to target a specific cell type. Explain the mechanism of how (from start to finish) these cells are engineered.

a. Collect white blood cells from patient
b. Isolate T cells
c. Engineer T cells to recognize cancer cells using CARs– insert CAR gene (chimeric antigen receptor)
d. Grow and proliferate modified T cells in culture
e. Infuse into patient
f. CAR T cells bind to cancer cells

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Name any 2 exo and endogenous DNA damaging agents

Endogenous agents
- material that is present and active in an individual organism or living cell but that originated outside that organism
- diet related (bile acids), Macrophage and neutrophil produced ROS

Exogenous agents
- smoke, radiation, genotoxic, inflammation

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Define ROS & RNS

ROS – Reactive Oxygen Species
- unstable molecule that contains oxygen that can easily reacts with other molecules

RNS – Reactive Nitrogen Species
- family of anti-microbial molecules

What are the primary outcomes of damaged DNA (cellular and molecular level in stem cells?

Overall Outcomes – Disease Pathogenesis, Increased Apoptosis, Increased Senescence

Molecular Outcomes – Genomic Instability, Epigenetic alterations, Dysfunctional telomeres

Cellular Outcomes – Stem-cell exhaustion, cellular senescence, inflammation

Briefly describe the diversity of stem cells and DNA damage in young and aged populations

Young – Healthy and asymptomatic

Old – More senescent cells with age and can become cancerous

Compare DNA repair in Stem Cells and Somatic Cells

Stem Cell – HDR, increase checkpoint response Somatic Cells – NHEJ, decrease checkpoint response

Define a stem cell niche. Mention any 5 signals that help in self renewal and differentiation of stem cells in their niche.

Local microenvironment of a stem cell & helps in interacting with each other b. Surrounding Cells c. Exosomes d. Lipids e. From the extracellular matrix

What is auto and paracrine signaling? Mention 4 causes of impaired stem cell self renewal

Autocrine – self signaling (self-renewal) Paracrine – from neighboring cells (growth factors) Causes i. Stem cell pool exhaustion ii. Unwanted differentiation iii. Reduced DNA repair – transform cells iv. Increased cell senescence

What are intrinsic and extrinsic factors and why are they important?

Intrinsic Factors – Post-translational Modification (add different functional groups like methyl), Micro- RNA Extrinsic Factors – New environment or lack of niche signals

What is the importance of a stem cell niche?

a. Protection of external environment (ex. Brain is protected by BBB) b. Controlled division c. Their own clone/colony of stem cells

5. How do bone marrow cells help in regulating HSCs? (SMART)

a. Self Renewal b. Maturation c. Apoptosis d. Resting e. Trafficking

Which cells surround Hematopoietic Stem Cells?

a. SNS Neurons b. Bone Marrow Macrophage c. Mesenchymal Stem Cells d. Bone Marrow Endothelial Cell

Describe how these cells help in HSC regulation in their niche: Osteoblasts, Osteoclasts, MSCs, Endothelial cells, Macrophages, Megakaryocytes, T-reg, Adipocytes

Osteoblasts a. Important cells to maintain HSC pool b. B-catenin, osteopontin Osteoclasts a. Induce osteoblast development b. Eat osteoblasts to remodel bone MSCs a. Self-renewal and maintenance b. IL-7 (growth and proliferation) Endothelial cell a. Self-renewal and maintenance, attachment b. E-selectins, CXCL-12 Macrophages a. Homing (attach) b. Integrin subunit alpha Megakaryocytes a. Inhibit proliferation b. TGF-B, thrombopoietin (CXCL 4 – blood clotting) T-regs a. Induce HSCs to differentiate to B cells (Ab secretion) & T cells (Th + Tc) Adipocytes a. Regulators of HSC differentiation (brown vs white fat cell balance)

Describe the mechanism of Jagged-1 on HSCs. Do you see symmetric or asymmetric HSC division?

HSC closer to Osteoblasts – self renewal by Jagged 1 (secreted by osteoblasts) HSC further away with osteoblasts – differentiation (No jagged 1 from osteoblasts)

What is a capillary and a sinusoid? Why are sinusoids important for HSCs?

Capillary – basement membrane is completely surrounding endothelial layer Sinusoid – lymph tissues - incomplete basement membrane (intracellular gaps - on purpose for interaction with immune cells – HSC)

Define Proteoglycans

Protein that provides hydration and swelling pressure to tissue

Define Polydendrocytes

NG2, population of CNS cells

Define Crypts of Leiberkuhn

Where intestinal stem cells are located

Define Paneth Cells

Secrete lysosomes to control bacterial proliferation

Define Gut Enterocytes

For nutrient absorption like ions, water, nutrients, vtamins

Name any 4 factors that can induce molecular aging in a mature brain

DNA damage, Inflammation, Cellular Stress, Epigenetics

What cells types can Bulge Stem Cells become?

Dermal, stem and sebaceous gland cells

Mention any 2 signals for NSC self-renewal by CSF, Ependymal cells, endothelium, astrocytes, neuroblasts

CSF i. VEGF, PDGF-AB Ependymal cells i. Notch1, Jagged1 Endothelium i. BMP, VEGF, Jagged1 Astrocytes i. Jagged1, FGF-2, Shh Neuroblasts i. GABA, Notch 1

Explain innate vs adaptive immunity

Innate – 1st line of defense (physical and chemical barriers) Adaptive – 2nd line of defense (B & T cells)

Name any 2 cell types and their functions of the lymphoid and myeloid lineages differentiated from pluri/multipotent stem cells

Macrophages i. Myeloid lineage ii. Identify and phagocytize B Lymphocyte i. Lymphoid Lineage ii. Produce Ab and stores memory

In a nutshell, explain the adaptive immune system (with diagram)

a. Macrophage detect pathogen & phagocytize b. Antigen peptides are then presented to Th and Tc cells c. Th activate B cells to secrete Ab d. Tc kill the cells directly by binding and killing them (granzyme to induce apoptosis)

Identify T-cell and B-cell induced diseases (choose from the given list)

CD 8+ (Tc) i. Rheumatoid arthritis ii. MS iii. Crohns iv. Psoriasis v. Hashimoto’s (thyroid) B Cells i. Lupus ii. Graves

Define Autologous

i. Same donor and recipient ii. BM Stem cells, Adipose Stem cells

Define Allogenic

i. Donor is different ii. UCMSCs

Define HLA

Human Leucocyte Antigen proteins on cells to let your I.S know which are self vs. non-self

What is MHC? How many main classes of MHC are present? Mention 2 cell types for each MHC class molecules.

Major histocompatibility complex MHC I i. found in all nucleated cells MHC II i. Dendritic cells, B cells & macrophage

Mention the subtypes of class 1 and class 2 MHC

CLASS I i. HLA A, B, C CLASS II i. HLA DRB1,3,4, HLA-DQA1, HLA-DQB1,HLA-DPA1, HLA-DPB1

Define microgravity

a. Condition where cells appear to be almost weightless b. Changes the gene expression in cells

Mention 5 ways of obtaining microgravity (mention the quality/conditions of microgravity and time felt by objects in microgravity).

a. Drop tower – excellent – 5-10 seconds b. Suborbital spaceflights – good – 5-10 seconds c. Parabolic Flight – good – 20 seconds d. Orbital – excellent – endless e. Artificial – good for bio sample – endless but not real conditions

Briefly explain RhoA and Rac1 dynamics under short and long exposure to ug and at 1G

Short Term i. RhoA – inhibited to adapt to changes in cell shape ii. Rac 1 – induces ROS which activates detachment Long Term i. RhoA and Rac 1 are inhibited & can activate apoptosis after detachment (Rac 1)