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BIOL311 > Stem Cells > Flashcards

Stem Cells Flashcards

1
Q

Overall Question

A

Given the fact that newly fertilized eggs which come from somatic cells generate a zygote and the zygote is able to differentiate into any cell type – why can the zygote differenate into a human AND can we make a somatic cell into a zygote.

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2
Q

Stem cells + faith (stem cell ethics)

A

Question – Is OK to use hESCs fro stem cell thearapy
- A cathlic person would say no because they beleive that life begins at conception

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3
Q

Story (about wife)

A

12 years ago → his wife and him were sitting outside their house → RVB was going to give a presentation to university
During this time RVB was beginning to be interested in Stem Cells → thought Stem cells would be approved by the FDA in the near future – thought that stem cells derived from blastocyte would be approved
His wife said that this was controversial AND not ethical → his response was would you use it to save one of our daughters
Wife said it wasn’t ethical

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4
Q

Controversy with Embryonic stem cells

A

Controversy = Is a blastoctye a life?

***Answer to question depends on your religion + depends on what country you live in

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5
Q

Dr. Alfred Cioffi

A

Cathloic priest with 2 PhDs

–> He took a class with RVB – he said that the pope thinks that hESCs will soon be used for stem cell therapy –> the pope needed priests with a terminal degree –> Cioffi went to get his PhD so that he could make decisions on how the church should preach about hESCs
- He needed to learn about hESCs so that the church would be able to tell people what to think if them
- He got his PhD in molecular biology at the age of 54 from Purdue

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6
Q

Uses of Stem cells

A

There are multiple uses for stem cells:
1. Increased understanding of how diseases develop
2. Use Stem cells to find a cure for diseases
3. Test new drugs for saftey
4. Generate new stem cells to replaice or aid diseased or damaged cells
5. Research How certain cells develope into cancer
6. Regernative medicine – Includes Tissue engirneered constructs + developing organoids
7. Fix genetic diseases
8. Clean Meat industry
9. Tissue engineering

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7
Q

Uses Stem cells as a cure (Ex.)

A
  1. Use stem cells to grow an ear
  2. Use stem cells to help with joint pain –> stop inflammation at the joint
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8
Q

What is included in Regenerative medicine?

A
  1. Tissue engirneed constructs
  2. Developing organoids
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9
Q

Stem cells + Clean meat industry

A

Using Stem cells and turning them into muscle rather than killing animals
- There are a lot of people investing in this
- Need to be able to make them profitable

Overall – Take Tissue from cow –> Get stem cells from tissue –> Differenate stem cells into muscle fibers –> make meat

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10
Q

Stem cells in Space

A

Nasa = 3D printing Biology research make the journey back to earth abord SpaceX’s dragon (NASA funded) – BioFabrication Facility

Issue – tissue engineering on earth has limitations because of gravity

Solution: Do 3-D printing in space

Example – Capillaries = hard to make on earth because of gravity –> they are printing capillaries using 3-D printing in space

***Moving towards printing human organs in space – The BioFabrication Facility (BFF) attempts to take the first steps toward printing human organs and tissues in microgravity using ultra-fine layers of bioink that may be several times smaller than the width of a human hair. This research is part of a long-term plan to manufacture entire human organs in space using refined biological 3D printing techniques.

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11
Q

Issue will Stem cells on earth

A

There are many things that we can’t do with stem cells on earth because of gravity – The biological printing of the tiny comeplex structure found inside human organs has been proven difficult under earth’s gravity
- Under earth’s gravity an initial scaffolding or support structure is needed to form the desired shape of the tissue

Example – Making capillaries –> this is an issue because need to feed tissue engineered constructs with blood

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12
Q

Helping the heart + stem cells

A

The Engineered Heart Tissues study looks at how human heart tissue functions in space – It uses unique 3D tissues made from heart cells derived from human induced Pluripotent Stem Cells (hiPSCs), essentially adult stem cells.
- Uses hiPSC – Looks at adult stem cells
- The engineered heart tissues = complex 3-D structures the size of a few grains of rice
- Tissues = more similar to issues in the i=body than flat cells in culture in a dish or in a flask

Overall – looking to see if making the engineered heart can be made better in space – is Tissue engineering better to be done in space because there is no gravity

***Heart made = VERY small

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13
Q

Stem cells + COVID

A

Usses stem cels to traet COVID – used Adult Meschymal Stem cells (AUTOLOGOUS)
- The MSCs = help the immune system by dampening the cytokin storm
- Using autologous adult adipose derived mesenchymal stem cells
- Providing immune support againt COVID
- They hope to limit disease progression and severity of covid – want to keep pateints out of the hospital and off ventilation

Bioformat FDA approved Phase II trail of Hope biosciences meschymal stem cells against covid
- Bioscineces = a clincal stage biotech company focused on developing cell based theraputucs for acute and chronic illness

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14
Q

Effect of adult meschymal Stem cells

A

They are immunomodulatory –> supress autoimmune disease
- They help hampen the cytokin storm
- Immunomodulatory + regenrative potential

Ex. Supress Rhumatoid Arthritus – in a recent clincal trial inflammation was reduced

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15
Q

What drives COVID disease progression

A

In COVID patients – inflammation is the driving force behind disease progression – it is critical to regulate the immune system as early as possible
- They are using stem cells in COVID pateints for their immunomodulatory effect –> It is hoped that the treatment will limit the progression and severity of COVID = keep them out of hospotal + off ventilation

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16
Q

Stem Cell

A

A cell that can either revew (divide) or differentate

***Which they do will depend on their niche

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17
Q

What controls the path a stem cell takes

A

The path the stem cell takes is controlled by the stem cell niche

***It is hard to know what controls stem cells in vivo

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18
Q

Stem cell niche

A

Includes Growth factors + ECM + enmvirnment

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19
Q

What affects the doublings in a stem cell

A

Influnced by:
1. The source (might depend on the age of the donor)
2. The type of stem cell

Example:
1. Adult stem cell (MSCs) – 50-100-200 doublings –> depends on the age of the donor (depends on the source)
- More like somatic cells
2. HESCs + iPSCs –> Immortal

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20
Q

Affect of patters of Stem Cell division

A

Different patterns of stem cell division creates a different proportion of cells and differentiated cells

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21
Q

Stem cell division

A

Stem cells can undergo asymetric divisions – one stem cell can me made into a new stem cell and one daughter cell becomes a differentiated cell
- Asymetric = means that the cell divides into two types of daughter cells

***This helps maintain the number of stem cells in the population

Some Stem cells can divide symetrically – done to increase the number of stem cells –> often done during development or during recovery from an injury

Some stem cells = can divide symetrically OR asymetrically –> one stem cell in the pool is diving symetricall and the other is not

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22
Q

Categories of stem cells

A
  1. Adult stem cells
  2. Fetal Stem cells
  3. Embryonic stem cells
  4. induced Pluripotent Stem cells
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23
Q

Adult stem cells

A
  • Most popular are adipose (fat) derived mesenchymal stem cells (adMSCs)
    - Now in more than 700 stem cell therapy trials globally
    - Includes Adult Mesenchymal stem cells – most commonly used for stem cells in Stem cell therapy
    - Can be Autologous

***In clinical trials

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24
Q

What are the most common cells used in Stem cell thearpy

A

Adult Mesenchymal Step cells – often derived from adipose tissue
***Cuurentley in clincal trials

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25
Q

Fetal Stem Cells

A

Includes:
1. Amniotic
2. Umbilical Cord
3. Placental

***They are not used often for stem cell therapy BUT umbilical cord is sometimes used

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26
Q

Embryonic Stem cells

A

Includes:
1. hESCs
2. hPSCs

***hESCs = in clinical trials as of 2010

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27
Q

Induced Pluripotent Stem cells clinical trials

A

Not in clinical trials in the US but patients are being treated in Japan + Australia
***Not in clinical trials in US but are in other parts of the world

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28
Q

Differentiation

A

Cells becomes more specialized such as a fibroblast or hepatocyte
- Occurs when a cell becomes more specialized

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29
Q

Fibroblast

A

Secretes Collegen

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30
Q

Hepatocyte

A

Used for Cytp450

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31
Q

Types of differentiation

A

Differentiation can be partial or full –> MEANS that accepted molecular metrics need to be used in order to compare the different stages of differentiation/level of differentiation

Example – need metrics to be able to compare one iPSC generated hepatocyte to another iPSC generated hepatocyte – because they might be different levels of differentiation

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32
Q

Progentitor cells

A

Stem cells that have “restricted lineage” – they are limited to only one or two types of cells that they can become

***Other cells = totipotent

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33
Q

Restricted Lineage Vs. Totipotent

A

restricted Lineage = can only become one or two cels types

Totipotent = can become all cell types

***Some stem cells = more restricted like progenitor cells and some are totipotent

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34
Q

Issue in stem cell differentiation

A

Issue = differentiation in stem cells is NOT all or nothing –> need metrics to know if stem cells are really differentiated (need metrics to know if a cell is fully differentiated)

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35
Q

Stemness of Stem cells

A

Not all stem cells are equal – some stem cells can make all types of cells BUT some have more restricted lineage –> some have less stemness
- Some might only be able to be one type of cell

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36
Q

Pathway from Stem cells –> restricted Lineage –> differentiated cells (Textbook)

A

Usually during each cell dividion of a multipotent somatic stem cell – at least one of the daughter cells becomes a stem cell like the paernt cell = stem cells undergo self renewal divisons so that you can maintain the number of stem cells in a person over time

VS.

Other daughter cells (Transit amplifying cells) – divide radpidly and only do a few number of self renewal divsions where they are similar to the parent stem cell –> INSTEAD they produce the lineage reistricted progentor cells –> THEN the lineage progentor cells divide and produce differenated cells

***The lineage progentor cells = can’t undergo self renewal –> they can only divide and differenate into cell types

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37
Q

Transit amplifying cells

A

Daughter cells of stem cells that divide rapidly and undergo limited number of self renewal divisions where they are similar to the parent cells –> THESE cells = ultimately produce the lineage restricted progenitor cells

***Overall – they produce the progenitor cells –> then the progenitor cells divide and make differentated cells

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38
Q

Transdifferentiation

A

aka “Direct Reprogramming” – differenated cells become another type of differenated cells without going thr the embryonic cell stage
- Abilitye of differenated cell to become another type of differenated cell without going through an embryonic step
- NOT LIKE iPSCs

Differentiated Cell type –> Differentiated Cell type

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39
Q

History of Transdififerenattion

A

First done experimentally in 1987 but several cells have been generated since then

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40
Q

Transdifferentiation in vivo vs. In vitro

A

In vivo – We do not think that it happens in vivo BUT we know that we can induce it in a lab
- It is really hard to figure out if it occurs in vivo

In Vitro – We know it can be done in a lab in vitro using Transcription factors + miRNA of different types
- Done using Transcription factors + miRNA of different types

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41
Q

Dedifferentiation and Redifferentiation

A

Ability of a cell to become more embryonic-like and differentiate into another cell type

Differentiated cell –> embryonic cell –> Differentiated cell

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42
Q

Reversine

A

Chemical that can induce dedifferentiation

***Chemical’s like reversine can induce dedifferentiation

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43
Q

Dedifferention/Redifferentation in vivo

A

We know it can be done ina lab BUT it is unlikley it occurs in humans
- Hard to know if it can occur in humans
- We know it can occur in animals BUT it does not occur in humans

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44
Q

Where is Dedifferenation found

A

Found in Red Spotted Newts

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45
Q

Red spotted Newts

A

They can go through dedifferentiation (can make new lens of eye + limbs)

  1. They can make new lens of eye using retinal pigment epithelial cells
  2. If you cut off one of their legs –> they can regernate a limb by dedifferentiated the cells that are left to be embyoic state and making the cells differentiate into leg cells
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46
Q

Department of defense + Red Spotted Newt

A

The department of defense was very interested in the newt differentiating –> they did a lot of research on the newt to tray and apply it to humans BUT they could not figure it out

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47
Q

Stem cell niche

A

aka “Stem cell microenvironment” – very complex

Includes:
1. Neighboring cells
2. ECM
3. Local Growth factors (FGF + others)
4. Physical environment (pH + Oxygen tension + pressure)

***part of the puzzle to solve what drives stem cell differentiation

***Includes everything – includes the immediate environment around the stem cells + includes Integrin receptors (affects stem cells)

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48
Q

Importance of Stem cell niche

A

Critical in controlling cell division vs. differentiation – control if cell will divide into stem cells or will differentiate into a more specific cell type

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49
Q

Potency

A

How many types of cells can the stem cells differentiate into

***We are able to classify stem cells based on potency

Types of Potency:
1. Totipotent
2. Pluripotent
3. Multipotent
4. Unipotent

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50
Q

Totipotent

A

Stem cell can differentiate into all cell types –> stem cell can generate all cell types

***Highest Level of Stemness

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51
Q

Pluripotent

A

Stem cell can divide into many cell types BUT not all
- Has more restricted stemness compared to totipotent

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52
Q

Multipotent

A

Stem cell that can generate several cell types
- Stemness is even more restricted than Pluripotent and Totipotent

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53
Q

Unipotent

A

Stem cell can only different into one cell type
- Differentiate into one cell type only

Example – Metaplicodese –> Blood cell generate

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54
Q

Blastocyst

A

Late pre-implanation stage embryo

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55
Q

Where do hESCs Orginate from

A

hESCs originate form inner mass of blastocyst

 - hESCs = made from a blastocyst
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56
Q

Question when generating hESCs

A

Question = are the stems cells derived from the blastocyst Totipotent

TO ANSWER –> USE Chimera Test

***NOTE – hESCs = made from the inner mass of blastocyst

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57
Q

Use of Chiemra Test

A

Chimera test = used to test/prove the totipotencey of stem cells
- Chimera test = the only true test of totipotencey of a candidate stem cell
- Asking if the stem cell is totipotent –> Need to see if it is able to make ALL cell types

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58
Q

Where can the chimera test be used

A

Chimera test = legal to be done in mice BUT not with humans

THIS MEANS –> we can beer prove that human stem cell derived or isolated on the lab is truly totipotent

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59
Q

Issue with proving human stem cells are Totipotent

A

You can’t do a chimera test in humans –> THUS we can never prove that human stem cell derived or islated in the lab is truly totipotent

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60
Q

Chimera test process

A

Implant the test stem cell in a blastocyst –> THEN implant the chimeric embryo into a surrogate mother –> Track the cell in all tissues and organs of newborn (can track because the stem cell was GFP labeled)

***Label the cells with GFP to be able to track the stem cells in the offspring mouse

RESULT: mESCs (mouse stem cells) = totipotent but we can’t say the same for hESCs (because can’t do the test in humans)

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61
Q

Chimera test process (Mine)

A

Take the cell stems –> ADD GFP to the cell –> Add the green stem cell to a blastocyste –> Put the now chimeric blastocyst into a surrogate mouse –> Get offspring mouse

Offspring = can have green spots OR it can be all green

   --> IF ALL GREEN = that means that the stem cells are totipotent --> they differentiated into all cell types

***You can cus the spotted green mouse open and look at the green cells to see all of the cell types that the stem cells were able to differenate into

**GFP added = constitutive marker –> makes the cell always green
**Chimera test = makes chimeric organisms

Results of mESCs: Mouse was fully green –> Shows that the mouse embryonic stem cells are totipotent

NOTE – we don’t know if hEScs are totipotent because we can’t do chimera test in humans – we could only know if hESCs are totipotent if we did chimera test in humans – we can’t say that hESCs will act the same as mESCs

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62
Q

Importance of Biodistribution + homing

A

Important in stem cells + Stem cell therapy

Issue: We don’t know how to track it

Question = DO the stem cells find a home where they should go once they are introduced? – We do not know the answer

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63
Q

Biodistribution/Homing

A

The ability for stem cells to find a “home” – ability of stem cells to find its target tissue
- When we introduce a stem cell into a person –> we don’t know where the stem cell goes

Question = DO the stem cells find a home where they should go? – We do not know the answer

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64
Q

GFP + Homing

A

We can’t use GFP label to track stem cell in human = can do not know where stem cells go in humans once they are introduced into a person

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65
Q

MSCs going to damaged site

A

We know that damaged or compromised tissues release factors that cause endogenous MSCs to home in damaged site – we know how endogenous stem cells find a home
- Bone marrow MSCs = go to site of injury and differentiate there

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66
Q

Heart transplant + Homing

A

Transplanted a XX heart into XY pateinet –> when doing an autopsy they found that the XX heart had XY cardiomnycyes (10% of heart cardiomycytes were XY NOT XX) –> THIS DEMONSTRATES ENDOGENOUS STEM CELL HOMING AND REPAIR
- We know that damaged tissues release factors that cause endogenous MSCs to home in damaged sites

Story – They transplanted a heart from a female kinto a male –> when they looked at the heart theer was some XY genotype –> this can only happen if the damaged cardaic tissue recurts stem cells – THIS happens through bone marrow MScs – the stem cells go to the site of injury –> the stem cells fin the heart as their home and differentiate there

67
Q

Homing in vivo vs Stem cell thearpy

A

We know that engenous MSCs go to sites of tissue damage because of factors released by the damaged tissue BUT we do not know if stem cells introduced into a patient using Stem cell therapy find a home/where they find that home

68
Q

iPSCs (overall)

A

Revolution in stem cell research

***NOT FDA approved for treatment

***iPSCs = can be many cell types BUT NOT totipotent

69
Q

Who got nobel prize for iPSCs

A

Sinya Yamanaka – got nobel prize for iPSCs (2012)

70
Q

FDA + iPSCs

A

iPSCs = NOT FDA approved fro treatment

71
Q

iPSCs + CRIPSR

A

Can use CRIPSR with iPSCs for genetic correction – NOT FDA approved

72
Q

iPSCs Process

A

Take Somatic cell –> Use iPSCs reprogramming factors –> Somatic cell now acts like embryonic cell –> Can differentiate
- Induces Pluropotencey

73
Q

Use of iPSCs

A

Make a human hepatocyte with defects and THEN test drugs on them

Take patient with liver disease –> Make their somatic cells into iPSCs –>

After iPSCs generation from patients you can:
1. Do gene correction –> take repaired iPSCs –> differentiate them into hepatocytes –> Transplant repaired hepatocytes to patients
2. Take the patients hepatocytes –> Do disease monitoring –> make the patient iPSCs into hepatocyte –> Test drugs on hepatocytes –> CAN then use the disease and patient specific drug to help patient

74
Q

STAP

A

Stimulus Triggered Aquition of Pluripotencey

75
Q

STAP (overall)

A

First reported in 2014 BUT is now retracted – was all fraud
**It was too good to be true
**Paper did the work right BUT the work was all fraud

Idea: Take somatic cell –> Add acid –> now have a stem cell that can differentae into other cells

76
Q

Work in STAP

A

They did all of the work right BUT it was all fraud

They did a Chimera Test BUT it was a little different

77
Q

STAP chimera test

A

They started with a GFP mouse (start = all green mouse) –> Then they isolated cells from mouse –> They used STAP to make cells into stem cells –> Put cells in blastocyst –> they put the blastocyst into a surrogate normal mouse

Result: The offspring of the normal mouse were all green = showed that the cells were totipotent
- Had GFP present in all tissue of offspring mouse = indicates that the STAP cells can turn into any cell/tissue in the mouse = cells were totipotent

78
Q

STAP Processes

A

Isolate cells –> Add acid –> Cells shrink –> Cells revert back to stem cell state –> Differentiate into many cells

79
Q

Fusogenic

A

Problem with stem cells = that the stem cells can spontaneously fuse with each other to form a tetraploid
- Can generate cancer stem cells

80
Q

Problem in stem cell therapy

A

Stem cells are fusogenic –> when injected into a patient mechanical stress can cause the stem cells to fuse with each other and form a tetraploid –> can generate cancer stem cells

81
Q

Membrane of Stem cells

A

Membrane = different –> the memebarnes can fuse together = get tetraploid cell –> unhappy cell that can release chromsome – can become oncogenic = big clincal issue

***Big problem in stem cell therapy = that stem cells are fusogenic

82
Q

Bio ethics

A

The norm of conduct

**Relative term
**Depends on your country – country dependent –> some things are legal in other countries that are not legal in the US

83
Q

Theraputive vs. reproductive cloning

A

Theraputic:
1. Not illegal in federal law
2. Done using SCNT
3. Used to treat Disease

Reporductive:
1. STILL Use SCNT
2. There is also no federal law against doing it in humans (no law because it hasn’t been done yet) – there are some state laws

Main difference:
In Theraputic = Goes to blastocyst BUT instead of putting the blastocyst in women you use the blastocyst for stem cell therapy

In reproductive = put the blastocyst in women

84
Q

Severse Combined Immunodeficiencey mice (SDIC)

A

Mice = have no immue system –> means that if you inject cancer cell or stem cell = the mouse won’t react
- Has no B or T cells –> thus have a compromised immune system

Use = to test for pluripotencey

85
Q

Use of SCID

A

Used to test for pluripotencey –> Put the stem cell in the mouse THEN see how the stem cell acts
- The stem cell is able to act because there is no immune system to fight it

***Used in cancer + stem cell research

  1. Used for determining if an injected candidate stem cell can differentiate in vivo into a multitude of types in vivo
  2. Used to determine if a candidate human cancer cell can generate tumors in vivo
86
Q

Generating Stem cells in a labratory

A

Three ways to generate stem cells in a lab:
1. SCNT
2. hPSC
3. iPSC

87
Q

SCNT – Somatic cell nuclear transfer

A
88
Q

SCNT History

A

Overall – has been around for a while (dates back to 1960s)

  1. John Gordon
  2. Sir Ian
89
Q

John Gordon

A

Was doing SCNT – Took intesitnal epithelial cell from a frog –> Did SCNT – removed an egg nuclues and put in somatic nuclus

Result: Got a tadpol

***First case of cloning BUT it wasn’t recognized until 2012 – in 2012 he was recognized and got a nobel prize

90
Q

Why was Gordon recoginzed when he was

A

He was recognized in 2012 because sir Ian did SCNT and got dolly at the same time

***Dolly = more known

91
Q

SCNT

A

Remove the egg nucleus and put in a somatic nucleus

***Enucleate egg – take out egg nuclues –> THEN add in somatic nucleus

***SCNT shows that a somatic nucleus can create an entire functioning animal due to cytoplasmic factors in egg

92
Q

Best source of stem cells for theraputic Purposes

A

SCNT – the best source of stem cells for theraputic puproses

  - Want a stem cell from own cell AND want to make the stem cell totipotent -- Take a human somatic cell --> Do SCNT --> take cells and intrdouce the SCNT cell and put it back into the person

***Would be autologous – would be a better autologous than MSCs

93
Q

Sir Ian Wilmut

A

Clones Dolly in 1996 BUT john Gordon did it with frogs in 1960 – John gordon got nobel prize in 2012

94
Q

Problem in SCNT

A

Very challenging – still very challenging

Very low efficiency –> 1000s SCNT are required for one implantable embryo

Because it is so difficult – many SCNT researchers left the field – very few people do SCNT now

***the efficiency of it working is very low = very hard to do = very few people do it

95
Q

First pet clones

A

Little nicky – 2004 (cat)

96
Q

Modern SCNT

A

Some are still attempting human SCNT designed for therapeutic cloning because hESCs can serve as an autograft

***SCNT is not illegal BUT is very hard to to

97
Q

Why can’t somatic nuclus serve as a nucleus of Zygote

A

Question in SCNT –> realized from SCNT that the cytoplasmic factors in the egg can convert a somatic nuclus so that it can make an organism

***Can use a somatic nuclus and make it differentate based on the niche

98
Q

DOLLY

A

Clones sheep using SCNT

- Dolly was born at Edinburgh University in 1996
  - Dolly was euthanized at age 6 due to lung disease and advanced arthritis but why?
 - But other identical clones, now 9 years old, from the same SCNT (Debbie, Denise, Diana and Daisy) are fine!
99
Q

Monkeys + cloning

A

Monkeys were clones in 2018 – first successful cloning of primates

***Primates have been cloned BUT congress still hasnt banned human cloning

100
Q

Pathogenesis

A

Making hPSCs

101
Q

hPSCs

A

hPSCs = not in clincal trials in the U.S BUT they are in clincal trials in other countries

***People want to use hPSCs for many clincal applications

102
Q

International Stem Cell Corporation

A

Famous for Pathogenesis
***They say that they are making stem cells with fewer ethical problems

103
Q

Nickmame for Pathogenesis

A

Virgin Birth –> because you do not need sperm

104
Q

History of Parthenogensis

A

Parthenogensis = was first shown in 1913 by Loeb using two experimental systems

105
Q

Loeb Experiments

A

He was demonstarting artifcial Parthenogenesis k

Experiments:
1. Unfertilized sea urchin eggs were induced to undergo parthenogenesis by changing the osmolarity of the surrounding medium
- Had a sea urchin egg –> Changes the osomalroty –> got an embryo –> Got an organsism
2. Unfertilized starfish eggs could do the same using dilute acid

106
Q

ISCO

A

Used as a source of stem cells for stem cell therapy –> Making hPSCs = using Parthegnogensis

107
Q

Parthonogensis Process (ISCO process)

A

Normal: Fertilize egg –> get blastocyte –> Can put blastocye in human and get baby OR use the blastocyte and make embryonic stem cells for therapy

ISCO: Trick the egg into fertlization – > Get Zygote –> Get blastocyte –> Get hPSCs

108
Q

How to “trick egg”

A
  1. Use ionmycin –> increase the calcium concentration = get division process without sperm cell
    • Uses ionmycin
    • Inomycin = Calcium Ionphore
  2. Use Puromycin – blocks protein synthesis
109
Q

Benefits of hPSCs

A
  1. Only 200 to 300 eggs would be required to generated hPSCs that could match anyone in the world
  2. Claims that for stem cells and stem cell therapy → they should have a perfect match for the entire world
110
Q

Limitations and Issues with hPSCs

A
  1. All alleles will be homozygous because of no sperm
  2. Not FDA approved in US
  3. Is it ethical to create a human embryo? – they claim that it is not an embryo because it is not being implanted
  4. Sperm is important → gives the otehr half of chromosomes
111
Q

Clues to get figure out iPSCs

A

Clues = came from reserach on embryonic master genes

Clue#1 – Somatic DNA can be conviced to go into embryonic stage – knew from SCNT
- Found in 1960s – doing SCNT
- Know from SCNT that there are factors to tell somatic nuclues to become a whole organism

Clue #2 – Embyonic master genes –> Knew about embyonic master genes
- Also knew about the embryonic master switch genes

112
Q

Use of embryonic master genes

A

Used as programming factors

113
Q

Embryonic Master genes

A

Oct 4 + SOX + MYC + KLF4 (Nonog)

All = activate genes for self-renewasl pluripotencey + Repress genes that induce specific differenation pathways

They active genes encoding proteins and micro-RNAs important for the proliferation and self-renewal of ES cells as well as to those of many genes that are silenced in undifferentiated ES cells and that encode proteins and micro-RNAs essential for the formation of many differentiated cell types

114
Q

iPSC process

A

Have pateint –> Take skin biopsy –> Add MYC + OCT4 + SOX2 + KLF4 (add 4 reproramming packaging repression factors) –> Get pateint sepcific iPSC – it becomes a clustering of embryonic bodies

After get iPSC:
1. Can repair the damage casuing mutating using CRIPR –> Then differentate the cells into healthy cells –> Then put healthy cells back into pateint

  1. Can differenate the cells –> screen the differentated cells with a drug –> Use the drug in the affected individual
115
Q

Use of iPSCs

A
  1. Can repair the damage casuing mutating using CRIPR –> Then differentate the cells into healthy cells –> Then put healthy cells back into pateint
  2. Can differenate the cells –> screen the differentated cells with a drug –> Use the drug in the affected individual
116
Q

Medical Application of iPSCs (Textbook)

A

Medical applications of iPS cells – In this example, the patient has a neurodegenerative disorder caused by abnormalities in certain nerve cells (neurons). Patient-specific iPS cells — in this case derived by recombinant expression of the four Yamanaka transcription factors in cells isolated from a skin biopsy — can be used in one of two ways. In cases in which the disease-causing mutation is known (e.g., familial Parkinson’s disease), gene targeting could be used to repair the DNA sequence (right). The gene-corrected patient-specific iPS cells would then undergo directed differentiation into the affected neuronal subtype (e.g., midbrain dopaminergic neurons) and be transplanted into the patient’s brain (to engraft the nigrostriatal axis). Alternatively, directed differentiation of the patient-specific iPS cells into the affected neuronal subtype (left) will allow the patient’s disease to be modeled in vitro, and potential drugs can be screened, aiding in the discovery of novel therapeutic compounds

117
Q

DATELINE – genes that convert human somatic cells to stem cells

A

Two group showed that somatic humans cells can be converted to true stem cells with 4 addiational genes

  1. Shinya Yamanaka of Kyoto University in Japan used a retrovirus to ferry into adult human fibroblasts – Genes = OCT3/4, SOX2, KLF4 and c-MYC genes.
    • Source were skin cells from a 36 YO and 69 YO (old cells)
  2. James Thomson of the University of Wisconsin (pioneer in hESCs) used OCT3/4, SOX2, NANOG and LIN28 genes
    - Worked with mouse embryonic stem cells

***The two groups worked together – they both had the same technology –> published paper together at the same time in the same journal

118
Q

Issue in iPSC research

A

When doing the research the researchers were using nuclei of old cells –> it would have been easier if they used young cells

119
Q

How do you prove that the cells are stem cells?

A
  1. Use RT-PCR
  2. Use SCID mouse test
120
Q

Use of RT-PCR

A

Used to show that the cells generated were stem cells

  - Showed cells before and after treatments  ***Shows true differentiation in culture as revealed by appropriate markers -- looks for markers that defined nuerons + cardiomycytes -- they could see the markers in RT-PCR
      - Looks for markers that show differenated 
      - ALSO looked for master genes

After treatment: Showed the prescence of genes that means cells are not differentiated

121
Q

Use of RT-PCR

A

Used to show that the cells generated were stem cells

  - Showed cells before and after treatments  ***Shows true differentiation in culture as revealed by appropriate markers -- looks for markers that defined nuerons + cardiomycytes -- they could see the markers in RT-PCR
      - Looks for markers that show differenated 
      - ALSO looked for master genes

After treatment: Showed the prescence of genes that means cells are not differentiated

Master genes – the master genes were in the undifferentiated BUT not there in the differentiated

122
Q

iPSC Mouse SCID test

A

Result: The iPSC formed a teratoma in mice – had tumor at the site of infection

- Showed that the iPScs can make differtaied cells -- because there were difefrenated cells in the teratoma that the iPScs induced when they wer einjected into the mouse 
  - Teratoma mouse from iPScs generated many cell types --> Shows that iPSCs can differentate into cell types
123
Q

Teratoma

A

Non-milignent mass of diferenated cells derived from iPSCs

124
Q

Teratocarcinoma

A

A malignant teratoma that originates from embryonic cells or stem cells
- Find differentdiated cvells from stem cells + find human like structures

***THIS IS THE REASON WHY iPSCs are not approved – because they can generate a teatocarcinoma

125
Q

Teratocarcinoma + other Stem cells

A

Can iPSCs and hESCs do this too? Are iPSCs in clinical trials like hESCs? (Answer: NO in US)

126
Q

Cellular Dyamins

A

Cell iPScs

127
Q

Promise of iPScs

A
  1. Basic research on differentiation
  2. Can make patient specific cells of individuals carrying genetic defects
  3. Source of cells in the future for stem cell therapy
  4. Will be useful in tissue engineering
128
Q

iPSC clincal trials

A

iPSCs = not approved by FDA in U.S. BUT Cynata Therapeutics (Australia) just completed (12/2018) first clinical trials in UK using iPSCs.

129
Q

Rejivinating old Fibroblast (DATELINE)

A

Scientists at the Babraham Institue have devleoped a method to “time jump” human skin cells by 30 years – turning back the aging clock

Called “Maturation Phase Transient reprogramming”

 - They measured telemere attrition + genetic instability + Epigenetic + transcriptional alterations + the accumilation of misfolded proteins (All of which are accepted markers of aging)

How did they do it??? – Used the same reprogramming factors as Yamanaka BUT instead of waiting the required 50 days they waited only 13 days –> the cell reversed its aging process by 30 years

**Normally it takes 50 days to culture iPSCs –> they asked what would happen if you don’t want 50 days –> they only wiated 13 days – they found that only waiting 13 days reversed the aging process
**May be used for cosmetic treatments

130
Q

Improving Liver generation in mice

A

The reserachers used iPScs BUT they only waiting 1 day instead of 5 days

result: The cells did not make teratocarcinomas

***Reserachers at the Salk lab used short-term Yamanaka factor protocll to partially reprogram mice love tissue
- The liver exhibited imporved regernation and younger charcahteriostics BUT they didn’t generate teratomas or other cancers that are typically developed with the standrad protocoll

***They are currentley trying to udnerstand the molcular basis of this rejuvination

131
Q

Pros and Cons of SCNTs vs. Parthenogensis Vs. iPSCs

A

SCNT
- Could be used for an autologous transplant if FDA approved
- No US federal laws banning therapeutic or reproductive cloning but some states forbid it.
CON –> But is it ethical? A “human embryo” is being created.
- Can’t use federal money
Parthenogenesis
- Can match to a world population – only 300 eggs required (?)
- Con – But all alleles are homozygous, not heterozygous
- Allogeneic, not autologous like SCNT unless female donated egg
- But is it ethical? A “human embryo” is being created.
iPSCs
- No “human embryo” created as in previous two
- Can be autologous or allogeneic
- CON - But potential for teratocarcinomas
- More pluripotent than fat (adipose)-derived adult mesenchymal stem cells and easier to procure

132
Q

Saftey Issues will Stem cells

A
  1. Tumorgenecity
  2. Immunogenecity
  3. Inappropriate differentation
133
Q

Stem Cells + Tumorgenecity

A

Stem cells have long telemers + can divide more times that normal cells
- They have a tendecey to form tumors + teratomas
- One clincal trial started in Japan at the RIKEN institue was stopped after one pateint due to concern

***Telemers = mitotic closk

134
Q

Stem cells + Immunogenecity

A

Stem cells = have a tendecey to trigger immune response
- Teh more frequent the stem cell injection the higher the chnace of immune rejection complications that can include anaphylaxis
- Auologouse + Allogeneic can launch an immune response

135
Q

Stem Cells + Inappropriate diffreentiation

A

Risk of stem cells differentating into cells that were not intented and are not native to the target organ

Ex – A women injected with MSCs near her eye ended up with bone tissue growing inside her eyelid

136
Q

iPSCs used to repair brain injuries

A

Done in mice – used iPScs to repair stroke damage in mice

Goal = repairing nuerological damage – transplanting neuronal stem cells into damaged or diseased areas of the brain has been touted as a potential therapeutic option, although it has been met with some difficulties. Yet now, investigators at Lund University in Sweden may have just found a path forward for using induced pluripotent stem (iPS) cell-derived cortical neurons for treating stroke-afflicted rats.

New article results – showed that the approach was able to restore mobility and sensation of touch by reprogramming human skin cells to become nerve cells, which were then transplanted into the rats’ brains

137
Q

3D printing personlized cardiac patches and heart

A

“human heart” has been made from iPSCs –> VERY small heart
- Done using 3-D bioprinting
- The heart had capalarries

Question = can they make a fully functioning full human heart

Result - World’s first vascularized human heart with atria and ventricles and patches – heart size of a rabbit heart
- Generated from iPSCs and donor’s own ECM that is processed into a personalized hydrogel making them biocompatible for implantation in the future

Next step – Train them to behave like a real heart. Cardiomyocytes beat but not in a coordinated fashion.

138
Q

Example of Cell differentiation + how it is controlled

A

Regeneration of intestinal epithelial cells

139
Q

Tracking the Generation of Intestinal epthithelial cells

A

Can track using H3 - Thiamine –> over time can see the labeled cells moving up

Result: Over time can see the cells moving up

H3 Thiamin – labels cells in the S phase

***Like a pulse chase experiment

140
Q

Regeneration of intestinal epithelial cells

A

Have differentating cells in the Crypt

  - In the crypt -- have differenated cells into many kinds of cells based on the niche
141
Q

Where is differentiation of Regeneration of intestinal epithelial cells

A

Have differentating cells in the Crypt

***The stem cells = located in the crypt –> They differenate and push up
- Differenate based on the niche

142
Q

Hematopoiesis

A

The generation of RBCs
- Making stem cells that have limited stemness – limited to RBCs

143
Q

SCID of Hematopoisis

A

When look at the SCID model – can see what the cells differentiate into

144
Q

Stem cell therapy + Hematopoeis

A

Stem cell therapy = starts with Hematopoesis because it starts with bone marrow transplants – replacing the stem cells that make RBCs in pateints

***Used to treat blood cancers

145
Q

Hematopoiesis (Textbook)

A

Early in mammalian development, multipotent hematopoietic stem cells often divide symmetrically to increase the numbers of stem cells. In adults, they generally divide asymmetrically to form one daughter cell that is multipotent, like the parent stem cell, and another daughter cell with a more restricted fate. Recent single-cell sequencing of purified progenitor populations revealed an unanticipated heterogeneity; there are at least 18 distinct subtypes of transit-amplifying hematopoietic progenitors. These multipotent transit amplifying cells are likely capable of limited numbers of self-renewal divisions but, depending on the types and amounts of cytokines present, they undergo rapid rounds of cell division and generate different types of progenitor cells (light green). These progenitors are either multipotent (e.g., bipotential granulocyte — monocyte and erythroid — megakaryocyte progenitors) or unipotent in that they can give rise to more than one type or only a single type of differentiated blood cells, respectively; they respond to one or a few specific cytokines. The lymphoid branch has not yet been studied in such great detail with single cell transcriptomics; future work should reveal more about how the B, NK, and T cells of the immune system (Chapter 24) are specified. Some of the cytokines that support this process are indicated

146
Q

Fluorescent Bar Coding

A

Sued a blood system –> Put into zebrafish BUT the blood was labeled with GFP – the cells were coded so that when they differenated they would all be diferent colors
- Different colors = all different kinds of cells
- sued flourescent protein probes

147
Q

Zebrabow

A

A specially bred (transgenic) zebrafish whose hematopoietic stem cells can fluoresce up to 80 different colors so that stem cell fate can be tracked
- Multiple copies of genes for red to blue fluorescent proteins are a permanent part of the genome of Zebrabow

148
Q

Ublical Cord blood

A

Cord blood = source of stem cells used in stem cell therapies

149
Q

Companies + Cord blood

A

There are companies that store cord blood
- Often store if the family has genetic disease or want as a source of cells later in time

150
Q

Types of Cord blood Banks

A

Private + Public

151
Q

Private Vs. Public cord blood banks

A

Private
1. Incorporated as a “for profit” organization
2. Donors pay an initial fee and a maintenance fee
3. Cells not available to the public
4. Better if there is a genetic disease in the family and multiple members require the cells
Public
1. Incorporated as a “not for profit” organization
2. Available to the public through the National Marrow Donor Program through which cord blood is matched

152
Q

Via Cord

A

Cord blood Bank
***RVB worked with them to make technology to store cells

153
Q

C.Elegans

A

Model organism to study cell differentiation

**Made by Robert (NOT RVB)

154
Q

Virtues of C. Elengans

A
  1. Easy to grow on agar plates
  2. Non pathogenic
  3. Translucent – can optically section through organism
  4. Stable mutant are available for study
  5. All cells have been coded and differentiation predicted
  6. Cell division/differentiation patterns can be predicted and always follow the same pattern
  7. Many genes like the apoptotic genes have mammalian homologs
  8. In fact, the apoptotic genes were first identified in C. elegans – Excellent model to study apoptosis
  9. First microRNA (miRNA) discovered (later slide)
155
Q

Stable mutants of C.Elengans

A

Have made stable mutants that don’t undergo apoptosis

156
Q

Genes in C.Elangans

A

It has many genes –> usually one cell goes to two daughter cells and one daughter cell goes to apoptosis– they made a mutant that doesn’t go through apoptosis – allowed them to find genes for apoptosis

157
Q

Use of C.Elengans

A

Can watch differentation – can track every cell
- Each cell can be given a code number –> since there are only 1000 cells this is manageable

158
Q

Question in C. Elangans development

A

Question – when and how is the organismic polarity established

159
Q

How is C.Elengans organismic polarity established?

A

Have Par proteins – Par proteins identofy the anterior form the protestori – they localize at different ends

Par proteins = establish polarity

160
Q

Par proteins (textbook)

A

Par proteins are asymmetrically localized by antagonistic interactions in the one-cell worm embryo. (a) DIC images of wild-type and par3 mutant embryos. In wild-type cells, the AB cell is larger than the P1 cell, whereas they are the same size in the par3 mutant. The par3 mutant also has a defect in spindle orientation (as seen by microtubule staining in green) and P-granule (red) segregation. DNA is stained blue. (b) Complementary localization of the anterior Par complex (Par3-Par6-aPKC) (red) and posterior determinants (green) in the one-cell embryo. (c) Antagonistic interactions between the anterior and posterior Par complexes. When Par3 becomes localized to the anterior it recruits the other members of the complex, Par6 and aPKC. The kinase activity of aPKC can phosphorylate local Par1, which inhibits its association with the anterior cortex. Par1 and Par2 associate with the posterior complex, and Par1 kinase can phosphorylate any Par3 to locally inhibit its association with the posterior cortex.

161
Q

miRNA + C.elegans

A

C.Elegans –> Heterochronic Mutant was Key to Discovering lin-4 RNA – the first microRNA (miRNA) ever discovered

Example mutant – miRNA discivered in C.e;egans model –> made a heterochronic mutant (has different timing) – mutant makes PDNB before it should

Track Lin 4

162
Q

Lin 4

A

The first miRNA found – unlike strands of mRNA it doesn’t code for proteins because it is double stranded = doesn’t code for a protein

163
Q

Xenobots

A

DATELINE –made the first reproducing stem cell fueled living Xenobots
- They used stem cells –> stem cells organized into moving entities – make Xenobots that can reproduce
- Discovered an entirley new form of biological reprdouction difefrent from any animal pr plant known to scinece

***May have therapeutic application

164
Q

New way of reproduction

A

Frogs have a way of reproducing that they normally use but when you … liberate the stem cells from the rest of the embryo and you give them a chance to figure out how to organize in a new environment, not only do they figure out a new way to move (below left), but they also figure out apparently a new way to reproduce by pushing cells together in a clump to make another C-shaped Xenobot (below right – green ball of cells)

BIOL311 (4 decks)

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