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Stem Cells > Midterm 2 Lec 13-23 > Flashcards

Midterm 2 Lec 13-23 Flashcards

1
Q

What are the two types of stem cells?

A
  • Hematopoietic stem cells: Red bone marrow (highly vascularized)
  • Mesenchyme stem cells: Yellow bone marrow
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2
Q

What is the role of blood vessels in bone marrow?

A

they prevent immature blood cells from leaving the bone marrow

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3
Q

What potency do hematopoietic stem cells have?

A
  • Multipotent
  • Have the potential to differentiate into any type of blood cell
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4
Q

What is the hematopoietic lineage? (What cells does it differentiate?)

A

HSC

1) → Mulitpotent progentitor cells

  2) → Lymphoid progenitor cells

        3) → Natural killer cells AND  → Lymphocytes (b cell and t cells) 

   2) → Myeloid progentitor cells   

         3) progenitor cells 

              4) → Erythrocytes (red blood cells) AND →Platelets 

         3) progenitor cells  

               4) → Leukocytes
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5
Q

What potency do mesenchyme stem cells have?

A

Multipotent

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6
Q

What are some cells types mesenchyme stem cells differentiate into?

A

(connective and some epithelial cells)

  • Bone
  • Fat
  • Muscle
  • blood vessels
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7
Q

Why is an important characteristic of HSC that make it an ideal candidate for various clinical trials?

A
  • their capacity to provide complete restoration of all blood cell lineages after bone marrow ablation
    • Respond to external environment/biological cues such as infection, therapeutic ablation, low oxygen, and loss of blood
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8
Q

Where does leukemia occur? (And what are the two characteristics/their names? )

A
  • Myeloid and lymphoid lineages
  • Acute Leukemia: Characterized by the rapid increase of immature blood cells
  • Chronic Leukemia: Distinguished by the excess build up of relatively mature but abnormal blood cells
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9
Q

What are the four types of Leukemia?

A
  • Acute Lymphoctic Leukemia
  • Chronic Lymphocyitc Leukemia
  • Acute Myeloid Leukemia
  • Chronic Myeloid Leukemia
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10
Q

Where are the stem cells in Hematopoietic cell transplantations collected from? 3

A
  • Bone marrow
  • Peripheral blood
  • Umbilical cord blood
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11
Q

What are the steps of bone marrow transplants? (4)

A
  • 1) Remove some bone marrow from the patient
  • 2) Give the patient immunorepressent drugs that will remove mutated HSCs in the patient (Immunoablation)
  • 3) Modify the removed HSCs to introduce the good copy of the gene
    • This can be done through Gene editing (CRISPR/CAS9 ) or Gene addition
  • 4) Transplant the modified HSCs back into the patient
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12
Q

What are the types of Bone marrow transplants? (4)

A
  • Autologous transplant: Patients receive their own stem cells taken from peripheral blood
  • Allogenic transplant: Patient receives matching stem cells from a close relative
    - Potential problem of immune rejection
  • Syngeneic transplant: Patient receives cells from an identical twin
  • Haploidentical transplant: Based on matching a donor with the patient via HLA matches
    - mismatch can lead to immune reaction
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13
Q

How can Hematopoietic stem cells be isolated?

A

FACS: Using specific antibodies that only bind to specific marker protiens

Hoechst dye: When the bone marrow is stained with dye a small side population has very low Hoechst fluorescent which are the HSCs
- because their ability to eflux or kick out the Hoechst dye with high efficiency due to the activity of the membrane pumps

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14
Q

What is sickle cell disease and what is it caused by?

A

It is a Monogenic disease (caused by a mutation in one gene) caused by the mutation in BOTH copies of the beta-globin gene (HB^B) that encodes for the hemaglobin protein which carries oxygen in red blood cells

Causes the RBCs to form an abnormal shape which makes the cells less functional
- They can’t carry oxygen as well and they are prone to clumping (can cause strokes)

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15
Q

What are the two types of transplants for sickle cell?

A

Autologous and allogeneic

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16
Q

What are 2 types of gene therapy?

A
  • Gene addition
  • Gene editing
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17
Q

What are the steps in gene therapy in autologous HSC treatment?

A

1) Remove HSCs from the patient’s bone marrow or blood

2) Have the patient undergo immunoablation (kill remaining HSCs and other blood cells that carry the mutation)

3) The HSCs that were removed will be modified in the lab using a gene therapy

- Gene editing: the HSCs are genetically edited using CRISPR/Cas 9 to fix the mutation
- Gene addition: a virus is used to introdice a functional copy of a gene in HSCs

4) The modified HSCs are transplanted back into the patient (via intravenous infusion)

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18
Q

What are the steps in allogeneic HSC treatment?

A

Transplant HSCs from another person that doesn’t have the mutation that causes sickle cell diease → So if the patient starts making normal RBCs from the transplanted HSCs the disease will be cured

It okay if there is a mix of patient and donor cells in sickle cell disease (Chimerism) as long as the majority are from the donor

1) Find a donor with matching Human Leukocyte Antigens (HLA) → They are proteins found on the surface of cells, which allow the immune system to recognize self

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19
Q

What are some potential problems with allogeneic HSC treatment?

A
  • Rejection of the transplant: The patient’s remaining immune system attacks the transplanted cells and they do NOT engraft ( divide and function in the patient)
  • Graft vs Host Disease (GVHD): (The transplanted HSCS make immune cells)The transplanted cells do NOT recognize the patient’s own cells as self and they attack them
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20
Q

What are savior siblings?

A
  • If a child need a HSC transplant the parents can create an exact HLA match through IVF
  • During the 8 cell stage, a cell can be removed and have a preimplantation genetic diagnosis to check that the HLA markers match and if they have the same genetic disease
  • Once the right embryo is chosen they let it form a blastocyst and is implanted and birthed
  • The HSCs are then removed from the umbilical cord and transplanted back into the child
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21
Q

What are the four principles of Bioethics? (4)

A
  • Justice
  • Respect for autonomy
  • Beneficence
  • Non- maleficence
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22
Q

What are some ethical issues involving savior siblings?

A
  • One child is prioritized over the other
  • Autonomy of the savior sibling
  • pre implantation diagnosis: Designer baby
  • does the savior sibling need to keep donating in the future
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23
Q

What is cloning?

A

Process of creating genetically identical tissues or individuals from a single donor

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24
Q

What technique is used for creating clone embryos? AND what are the who types of cloning and their steps ?

A
  • Somatic Cell Nuclear Transfer (SCNT)
  • Somatic Cells → SCNT → cloned embryos
    • 1) → Therapeutic Cloning (autologous treatment) → isolate ntESC (creates new embryonic stem cell line with the same genetic material as the donor)
      • iPSCs are more likely to be used for autologous transplants because they are less controversial
    • 2) →Reproductive cloning → Implant embryo into uterus → clone individual
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25
Q

What are the steps of SCNT?

A

1) enucleation of oocyte (remove the nucleus with all its DNA)
2) Transfer a nucleus (or a cell that contains the nucleus) into the enucleated oocyte
- At this point it is NOT active and has two copies of a genome
- Normally initiated by a sperm cell
3) Activate the oocyte to get it to start dividing and make and embryo
- electric shock or drugs can be used

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26
Q

What were the steps taken to clone Dolly the sheep? Who would Dolly the sheep look like?

A
  • 3 sheep were used in the process
  • Sheep A donates the oocyte
    - The oocyte is enucleated (remove nucleus)
  • Sheep B Somatic cell donor → Contains 2 copies of genome 2n
  • The oocyte and somatic cell are fused together w/ electric shock → Cell is active → develops in vitro until blastocyst → Implant into sheep C
  • Sheep C: Surrogate → Births Dolly the Sheep

→ Dolly the sheep is a clone of sheep B

27
Q

What two biological facts were demonstrated in early cloning experiments?

A

1) Cloning an adult cell nucleus proves that all the genes necessary for development are still in adult cell → All cells contain the same genes

2) The oocyte must contain factors that allow for reprograming of the somatic cell DNA

28
Q

What is the difference between iPSC and SCNT gene expression?

A
  • In iPSCc we have to force expression of a few stemness transcription factors
  • In SCNT the oocyte does the job of reprograming the differentiates cell nucleus
    - The right stemness TFs and pioneer factors are in the oocyte cytoplasm
29
Q

Why is SCNT so inefficient?

A
  • Cloned embryos have a hard time developing extra-embryonic tissue (like the placenta)
  • the correct stemness genes aren’t all expressed
  • Some of the chromatin is resistant to reprogramming (stay in closed state)
    - Due to the way histones can be chemically modified to make them more likely to stay compacted or open up → Epigenetic memory
30
Q

What is the process of retrieving Embryonic stem cells?

A

sperm + oocyte → IVF → Zygote → embryo → ESC

31
Q

What is the process of induced pluripotent stem cells?

A

somatic cells → reprogramming factors → iPSC

32
Q

What is the process creating cloned embryonic stem cells?

A

Somatic cells → Nucleus SCNT → enucleated Oocyte → cloned oocyte → embryo ntESC

33
Q

How are stem ness and differentiation genes expressed in somatic cells and cloned ntESC?

A

Somatic sells
- Stemness genes: decrease in expression (Closed chromatin)
- differentiation genes: increased expression (open chromatin)

Cloned ntESC
- Stemness genes: mostly open/ expressed (not all genes are expressed)
- differentiation genes: Mostly closed But not all (some genes still expressed)

→ Epigenetic memory

34
Q

What are the steps to cloning a dog?

A

Dog A- (want to clone) remove a sample tissue
- somatic cells (2n)

Dog B- Oocyte donor
- Enucleate oocyte (remove nucleus)

→ SCNT: Fuse the oocyte and cell → Cloned zygote → embryo

Dog C- surrogate
- Implant they embryo into dog c
- Cloned Dog A

35
Q

What are some limitations/ ethical issues when cloning a dog?

A
  • Clone Dog A may not act like the original dog
  • Treatment of dogs in the company
  • Genetic illnesses may be in the clone
36
Q

What is necessary for de-extinction using SCNT?

A

1) Genetic material (preserved tissue of extinct animal)

2) Oocyte (can be from a living, close relative)

3) Surrogate (can be living, close relative)

37
Q

What was the process of de-extinction of the Pyrenean ibex?

A
  • A tissue sample is stores
  • enucleate a goat oocyte
  • create embryo
  • implant in a goat/spanish ibex (hybrid)
  • 1 successful pregnancy (let the pregnancy go for 162 days)
  • The goat was birthed via c-section and was only alive for 10 minutes

→ possible problem: how long should they let the gestation part go?

38
Q

What are some limitations/ ethical issues of de-extinction?

A
  • There is a lack of genetic diversity
  • we don’t know the consequences of reintroducing them to an environment
  • Will they go extinct again?
  • instead stay focus on endangered animals (this might cause complacency if we can bring any animals back)
39
Q

How does mitochondrial replacement therapy occur? How many peoples’ DNA is present?

A
  • oocyte donor → enucleate→ transfer nucleus from the mother’s oocyte → IVF → zygote will have some genetic material from the oocyte donor
  • 3 people
40
Q

What are three ways we can make new human tissues and organs?

A
  • Bioengineering
  • Organoid (in vitro, 3D culture)
  • Grow in animals
41
Q

Why are human organs grown in animals? Why use pigs?

A
  • There are many health problems that can be treated with organ transplants but there is a shortage of donated organs
  • Rather than making organs in a lab, the growing organs would receive normal growth signals from the rest of the body
42
Q

How do you use a pig to make a pancreas?/ What is interspecies blastocyst complementation?

A
  • Interspecies chimera: When human pluripotent stem cells are injected into a pig blastocyst

1) Use CRISPR/Cas9 editing in the pig zygote →Create an “empty niche” this can be done by deleting Pdx 1 which is a transcription factor that turns on the expression of genes that are important for pancreas development

2) Interspecies blastocyst complementation → Add human iPSC which have a functional copy of Pdx 1 into a pig blastocyst (this is important because the pig will only survive if the human cells fill the empty niche and make a functional organ)

3) Implant the blastocyst into a surrogate pig

4) If the human iPSCs don’t make a pancreas then the pig will die, If they do the pig will have a human pancreas

43
Q

How far has research progressed in this field? How have rats and mice been used?

A
  • Pdx 1 mutant pigs have been made and stem cells from another non mutant pig can be used to make a new pancreas in the mutant pig
  • Human iPSCs have been injected into pig blastocyst and after a month some of the human cells were found dispersed around the embryo in low numbers
  • A mouse pancreas has been grown in a rat and then pancreatic glands were transplanted back into the mouse. The transplanted tissue was functional
44
Q

What are some limitations and ethical issues

A
  • Species barrier & difference in developmental timing- pigs and humans are not that closely related and embryo development is different between the two
  • Animal rights issue
  • Religious issues
  • What if they gain human conscious
  • Human cells in the pig reproductive system
45
Q

What is an organoid?

A

made from stem/progenitor cells without a rigid scaffold that self assembles into small 3D structures reminiscent of developed organs

46
Q

How do you grow neural stem cells in a 2D culture? and the imitations

A

1) Start with Pluripotent stem cells (ESCs or iPSCs)

2) Allow the cells to aggregate into neural rosetta made up of nueroepithelial cells (multipotent stem cells in the nervous system) 

3) add nervous cells → Differentiate nueroepithelial cells into neurons

LIMITATIONS
- flat structure
- does not differentiate into all cells → limited types of neuron and glia cells

47
Q

Why are orgnaoids more useful that 2D cultures?

A

⇒ Organoids have an advantage over 2D cultures because they have more similar architecture to brain tissue and a greater diversity of cell types

48
Q

How are cerebral organoids made?

A

1) Start with hiPSCs and let them aggregate into embryoid body

2) add signals to induce pluripotent cells to become neuroepithelial cells

3) transfer the neuroepithelial cells into matrigel

4) Grown in a bioreactor (has cell culture media and signals)

5) stem cells will differentiate into variety of neurons and glia cells

  • Cells self organize into tiny brain
  • fold like brain cortex → cells form layers like human brain, hollow region w/ spinal fluid
  • cytoarchitecture: similar to human brain

→ Limiting factor: stay small because they don’t have blood vessels

49
Q

What are the applications of cerebral organoids?

A
  • study neural development → diseases that affect development
  • see how different organoids connect w/ each other
  • disease modeling
  • virus effects
50
Q

What are some ethical issues associated with cerebral organoids?

A
  • Are they conscious? NO
  • consent of patient
  • placing human cerebral organoids into animals
51
Q

What is neurogenesis?

A

the process by which new neurons are formed in the brain

52
Q

What are the two stem cell niches in the adult brain? where are they found? What is their purpose?

A

subventricular zone
- found under the lateral ventricle (fluid filled cavity)
- NSPCs differentiate into immature neurons and then migrate to the olfactory bulb where they mature
- We don’t exactly know their purpose for making more neurons in the olfactory bulb (receive info from the nose about odors)

Dentate gyrus in hippocampus
- found in the hippocampus (important to the formation of some long term memories and especially spatial navigation)
- Neurogenesis occurs in the dentate gyrus and the new neurons stay there and grow their axons to other regions of the hippocampus

⇒ Have the right microenvironment to keep the Neural Stem/Progenitor Cells undifferentiated

⇒ Stem cell niches are highly vascularized

53
Q

What are some things that increase and decrease neurogenesis?

A

Increase
- environmental enrichment
- exercise
- learning

Decrease
- chronic stress
- decrease in sleep
- increase in fat in diet
- alcohol and drugs

54
Q

What are two experiments to check that older humans can make new neurons?

A

BrdU labeling
- Cancer patients that were about to die were injected with a chemical called BrdU that gets incorporated into the DNA of dividing cells
- Once the patients dies researchers were ableto look in the dentate gyrus and SVZ and saw that some neurons had BrdU labeling
- shows that the neurons were made in the patient recently

Dex labeling
- label for immature neurons which express Dex
- use antibodies that bind with Dex to see if there are immature neurons in postmortem brain tissue

55
Q

Why can’t neurogenesis stop/prevent neurodegenerative diseases?

A
  • Rate of neurogenesis is low compared to the neurons dying
  • only make new neurons in two places (SVZ and Hippocampus)
  • disease often happen later/ the rate of neurogenesis decreases with age and with disease
56
Q

Which neurological diseases would be best treated with stem cell therapy?

A
  • easiest to treat a disease that has one specific area with damaged or degenerated neurons
  • if the loss is systemic (throughout the body) it’s more difficult to treat with a transplant

→ Neurodegenerative disease: Occur later in life and involve a progressive loss of neurons in the brain, often focused on one region

→ Acute damage: loss of neurons in one region all one once (EX: stroke, spinal injury)

57
Q

What cells are used for transplantation to treat neurological diseases? And which could be used for autologous/ allogenic transplants?

A

Stem cells sources
- Embryonic stem cells (Allogenic)
- induced pluripotent stem cells (Autologous)
- mesenchyme stem cells (from bone marrow or fat) (Autologous)

58
Q

What is cell substitution and when is it a good strategy?

A

good strategy if you’re treating a neurodegenerative disease that causes loss of one type of neuron in one region of the brain

59
Q

What is trophic support and when is it a good strategy?

A
  • For injuries that involve loss of multiple cell types → best to transplant stem cells or progenitor cells directly
  • some cells might differentiate but what seems more helpful are the signals that are released by the transplanted cells
  • signals decrease inflammation in the brain, improve survival of remaining cells in the area and increase neurogenesis
60
Q

What cells are commonly used in stem cell therapy experiments for trophic support? Why?

A
  • mesenchyme stem cells
  • They are abundant in the patient’s bone marrow and fat so it’s an autologous transplant
  • a trait of mesenchyme stem cells is that they migrate toward injured areas so they don’t have to be transplanted directly onto the injury site
61
Q

What happens during a spinal cord injury? What is a possible treatment?

A

During the injury axons are destroyed which extend up and down the spinal cord → can cause paralysis and loss of touch/pain sensation
- Axons in the spinal cord can not regrow but they can regenerate if stem cells (like MSCs) were transplanted nearby which release growth factors and decrease inflammation TROPHIC SUPPORT

The treatment seems safe (no teratoma) and sometimes it works and sometimes it doesn’t

62
Q

What can cause the different results in using stem cells to treat spinal cord injuries?

A
  • Differences in how the stem cells are obtained, grown or prepared
  • where the stem cells are transplanted
  • the type of injury and how extensive it is
  • the time between the injury and treatment
63
Q

What is Parkinson’s Disease? What is a possible treatment?

A

PD is a degenerative disease in which dopamine neurons die off → Causes motor impairments

1) Make iPSCs from donated fibroblast

2) Differentiate the cells into dopamine neurons

3) When the human dopamine neurons were transplanted into a monkey with PD it grew human neurons and improved motor function was seen

4) In clinical trials → if transplanted back into patient they can be autologous and allogenic

Stem Cells (3 decks)

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