mcb 38 midterm 2

List the two bone marrow types and the cells they differentiate into

- Hematopoietic: blood cells (lymphocytes, erythrocytes, leukocytes)

- Mesenchymal: bone, muscle, fat

Where are hematopoitec stems produced? What type of potency?

Red marrow of the bone. Multipotent

Name the two lineages hematopoietic stem cells seperate into. What are each of their function?

- Lymphoid progenitor cells: creates specific immune responses with antibodies

- Myeloid progenitor cells: produce RBCs and immune cells that attack

How do we identify and isolate hematopoitec stem cells?

- Experssion of CD133

- Lack of expression of cell surface markers

- lack of Hoechst dye

What role does Hoechst dye play in isolation of HSC? Where does it bind?

- Cells with potent hematopoietic activity efflux dye

- Binds in DNA

Autologous vs. Allogeneic bone marrow transplant

- Autologous: patients recieve own stem cells from peripheral blood

- Allogeneic: patients receive cells from close relatives or close HLA match

Allogenic transplant steps

- find donor with matching HLA markers

- immunoablation

- trasnsplant new stem cells

- give immunosuppresssants

Autogeneic transplantation

- remove HSCs from patient

- infect HSCs with virus that carries good copy of gene

- transplant modified HSCs back to patient

What are hyman leukocyte antigens (HLA)? Why pay attention to them in transplants?

- proteins on surface of cells for immune system to recognize

- highly polymorphic, so it can lead to immune rejection

What is a savior sibling?

- sibling born to provide a organ/tissue

How are savior siblings produced?

- IVF, pre-implantation genetic diagnosis (take on cell out of morula), matcha HLA, implant in mother,

- when child is born, we can take stem cells from umblical cord or bone marrow

4 principles of bioethics. Which ones are violated for savior siblings?

- autonomy, beneficence, non-maleficence, justice

- VIOLATED: autonomy, non-maleficence

What is cloning and the technique to carry it out?

- process of creating a genetically idenical tissue from a single donor

- Somatic cell nuclear transfer (SCNT)

Reproductive vs therapeutic cloning

- Reproductive: creating another organism

- Therapeutic: generate cloned ESCs to create tissues

What is the alternative to using therapeutic cloning?

- iPSCs, becuase they are less controversial

Why is cloning inefficient?

- uses old cells, which may have abnormalities

- they also have epigenetic memory

Explain epigenetic memory

- not all the stemness genes will be expressed properly

- some stay closed chromatin, some will open

Applications for reproductive and therapeutic cloning

- cloning pets or livestock: bring back dead pet

- de-extinction: bring back extinct animals

- autologous transplants: ntESCs

What is necessary for de-extinction using SCNT?

- intanct somatic cell nucleus of extinct animal

- egg/oocyte from related animal

- surrogate to carry out the embryo

Explain mitochondrial replacement therapy (MRT)

- help women produce a child without mitochondrial disease

- mtDNA comes from oocyte and everyone has same mtDNA as their mother

Steps of MRT

- remove nucleus of donor oocyte, transfer nucleus of mother's oocyte to donor oocyte, IVF

How can you grow a human organ in a pig?

- Interspecies blastocyst complementation

- Put iPSCs into pig blastocyst, then the embryo will have a mutation that will prevent developmentof an organ.

- Creates "empty niche" for human cells to fill

Scaffold

- 3D template that mimics ECM

Bioreactor

-Device/system that appllies different types of mechanical stimuli to cells

- allows for 3D culture conditions

Biodegradability

Ability to allow the body's own cells to replace the implanted scaffold and not release harmful metabolites

Biocompatability

- Ability to support cellular activity in order to optimize tissue regeneration without immune response

Pores

- Ensure cellular penetration and adequate diffusion of nutrients to cells

Decellularization

- Use of agents to remove cells with specific HLA markers while retaining ECM

Vascularization

- Body develops capillaries

- Major issue in bioengineering

Ceramics and their pros and cons

- Material with high mechanical stiffness, very low elasticity, and very hard brittle surface

- Pros: rigid, low elsaticity, bone microenvironments

- Cons: brittle, difficult to shape

Synthetic polymers, and their pros and cons

- Materials able to be fabricated with a tailored archituecture and controlled degradation

- Cons: risk for rejection due to bioactivity, and degradation can decrease pH and kill cells

Natural polymers and their pros and cons

- Materials that are biologically active

- Good for cell adhesion and growth

- Cons: hard to make scaffolds with homogenous structure, they have poor mechanical properties

Biological and ethical issues related to bioengineering tissues

- Biological: hard to scale up and mare large organs, grow blood vessels, make tissues with multiple cell types

- Ethical: how do you regulate them? Cost and access?

Organoid

- 3D cell cultures that incorporate key features of the represented organ

- Grown from stem and progenitor cells in vitro

Growing cerebral organoids in 2D

- Start with pluripotent stem cells and let them aggregate into embryoid bodies

- Forms embryoid bodies that will differentiate into neuroepithelial cells (multipotent)

- Will then become neural rosetts

- Add signals, they will become neurons and glial cells

What are limitations to growing neurons in 2D

- limited in the types of neurons you can make, since not like 3D brain

What step can you add to grow cerebral organoids in 3D?

- place neural stem cells into Matrigel

- Then grow partially-differentiated cells in a bioreactor with signals to ensure differentiation

Name one reason why cerebral organoids are not conscious and one evidence why they are conscious

- Not conscious: too small and simple and lacks rest of nervous system

- Conscious: can make synaptic connections with neurons in a mouse, synchronized electrical activity

Neurogenesis

- process of making new neurons from stem/progenitor cells

What are the 2 niches for neural progenitor/stem cells?

- Dentate gyrus

- SVZ

What happens at the olfactory bulb for neural cell development?

- immature cells from SVZ mature into full neuron and forms synapses with other cels

Positive and negative regulator of neurogenesis

- Positive: environmental enrichment, voluntary physical exercise

- Negative: stress, alcohol/drugs, sleep deprivation

Evidence that there is neurogenesis in human brains

- BrdU is seen in cells that have recently been divided. We see this in post-mortem brains

- Dcx is expressed in neurons post-mortem (only newly made neurons express Dcx)

Cell substituion

- uses differentiated cells

- differentiated transplanted cells with replace cells that are injured

- ex: parkinson's disease (one type of neuron died)

Trophic support

- stem cells (like MSCs)

- Stem cells will release growth factors and signals to help injured cells survive and decrease inflammation

- Stroke or spinal cord injury (multiple neuron types died)

Chimerism

• a mix of patient and donor cells

• could cause immune response

How to make iPSCs

• use skin fibroblasts

• introduce reprogramming factors

• use a virus to deliver the factors and revert them to a pluripotent state.

Hemapoietic stem cell characteristics

• red bone marrow
  - highly vascularized
  - considered rare
  - multipotent
  - multilineage
  - leukocytes, lymphocytes, erythrocytes

mesenchyme stem cell characterisics

• yellow bone marrow

• not very vascularized

• multipotent

• bone, muscle, fat

graft vs host

• donor HSC cells attack patient cells

What does sox2 do for fibroblasts?

• opens up fibroblasts

• binds to DNA

• binds to coactivator

• allows stemness genes to be expressed

• positive feedback: cells start to express own sox2 and oct4

Uses of iPSC

• genetically modify to treat patient

• study cellular malfunction

What do you need to consider for organ transplants?

• allogenic

• organ donors

• stability/suitability of organ

• not all parts are donatble

dentate gyrus

• new neurons stay here, axions are grown elsewhere

• part of the hippocampus

steps in bioengineering a tissue

• choose scaffold

• add stem cells or differentiated cells

• implant

• scaffold degrades

BrdU

• dye enters DNA of NSPC, which differentiates into a mature neuron with dye

• dye is found in SVZ and hippocampus

Cerebral organoid pros and cons

pros: cytoarchitecture (similar structure), less restricted, increased diversity

cons: no blood vessels, cells inside dont receive nutrients

strategies to form 3D scaffold

• inject cell into hydrogel

• allow cells to mirgrate into scaffold through pores

• decellulaization: enzyme digest cells and scaffold is left, repopulate with stem cells

Scaffold requirements

• Biocompatibility

  • Cells adhere/migrate through scaffolds. No immune response when implanted

• Biodegradability

  • Not a permanent implant. Can be rapidly cleared from body while tissue forms

• Mechanical properties

  • Balance between mechanical properties and porous architecture

• Scaffold architecture

  • High enough porosity to ensure cell penetration, nutrient diffusion, and waste removal. 

  • Ex: collagen works because of Arg-Gly-Asp AA sequence

• Manufacturing technology

  • Cost effectiveness and capability to scale scaffold production

types of tissue engineering

• 2D: skin cells, no blood vessels

• hollow tubes

• soft tissue

• dispersed group of cells

• rigid tissue