1 Reg Medcine

What are the 5 main areas of regenerative medicine

Cell and gene therapies
Stimulation of endogenous repair
Human tissue transplant
Xenotransplantation
Engineered tissues

What is the key aim of regenerative medicine?

• to replace, repair or regenerate cells, tissue and organs in order to restore health

• Translational science and engineering

Define stem cell?

unspecialized cells which have the ability to self-renew, producing more stem cells, and differentiate into other cell types

What are 3 types of stem cell used?

• Embryonic stem cells

• Induced pluripotent stem cells

• Adult (somatic/tissue) stem cells

How are stem cells detected?

dentified by the expression or absence of a number of transcription factors and cell surface markers

What can stem cells markers be used for?

enrich stem cell populations for further study or biomedical applications such as tissue engineering

What techniques can be used to determine expression of stem cell?

Western Blotting
RT-PCR
Immunocytochemistry
Flow cytometry

Define enriching cell?

isolate cells from other contaminating cells

Describe density centrifugation when collecting stem cells?

• Stem cells are in the mononuclear cell layer

• Can use tetrameric antibody complexes

  • Binding to unwanted cells and to red blood cells for an aggregated mass

Describe magnetic activated cell sorting

• antigen that recognises an antigen on cells of interest or on the unwanted cells

• Antibody has a magnetic nanoparticle attached to it

• Pass through a column with a magnet

• The cells with the tagging get stuck in the magnetic fields where the cells of interest pass straight through

• Take away magnet

• Flush away cells and collect in tube

What can flow cytometry be used for?

both analysing and enriching
for detecting specific molecules on and within cells

Describe the general principle of Flow Cytometry?

• cell suspension that passes very fast one by one in laminar flow through the laser

• Get various scattering of the laser beam depending on which cells are passing through

What can Flow Cytometry be used for in medicine?

immunophenotyping, diagnosis, cell sorting

What does the front scatter detector detect?

• Detecting the dimming of as the cell passes through

• Longer the dimming of life occurs for the bigger the cell

What does the side scatter detector detect?

• Detecting light bouncing of organelles and granules within the cell

• More side scatter more granular the cell is

Flow cytometry with antigens?

• Incubate cell population with these antibodies

• Antibody binds and cells with fluoresce a particular colour

• Can label cells with multiple antigens at anyone time

• permeabilise the cells to look ate antigens within the cell

• Can do up to 18 flus FSC and SSC

How are fluorophores of different antigens separated

• separated off by dichroic mirrors

  • Reflect certain wavelengths of light and let others pass

  • Direct the different colours of light into different detectors

What is intracellular antigen flow cytometery manily used for?

proteins, but DNA and RNA can also be detected

How does fluorescent-activated cell sorting work?

The computer gives the cells charge depending on the coloured label and the cells are separated this way

In Embryonic Stem Cells (ESCs) how long is the inner cell mass totipotent for?

• day 4 or 5

• From day 5 or 6 the inner cell mass within the blastocyst is pluripotent

  • These are cells that will go on to produce the adult organism

What are the master regulators of pluripotency?

all transcription factors

• OCT-4

• SOX-2

• NANOG

What are the complex set of interactions made-up of that regulate pluripotency?

extracellular signals, signalling pathways, transcription factors, micro RNAs and epigenetic factors

How are iPSCs produced?

• Transfection of fibroblasts with combinations of 24 genes known to be important in ESvery similar to ESC in terms of morphology, unlimited proliferation, surface markers, gene expression

• Differentiate into cells of all three germ layers in vitro and in teratomas

What can human induced PSCs be used for?

• Specific to the patient

• Used to make disease models

• Drug testing to identify new drug targets

• Can be used to correct genes implant back into patient

• Can be differentiated into cells of interest → infused back into the patient

• Can be used to generate engineered tissues

• Can differentiate into other stem cells

What is a stem cell niche?

• the microenvironment

• Niche regulates function

What type of interactions happen in the stem cell niche?

• by a combination of cell cell interactions

• Cell matrix interactions

• Soluble factors → growth factors small molecules

• Oxygen gradients

• Mechanical forces

Why are stem cell niches important?

To get the cells to differentiate into the type of cell we are aiming for

What can influence the type of stem cell produced?

mechanical properties of the growth surface
promote self-renewal or influence lineage

Where have hPSC tried to be implemented?

Development of mature β cells from hPSCs → treated signalling in the development of beta like cells
Parkinson’s Disease → replace midbrain dopaminergic (mDA) neurons to replace those lost in PD using patients own cells

Why do we not inject IPSC?

only injected differentiated cells to ensure no formation or teratoma
by ensuring no expression of pluripotency markers

What type of therapy could IPCs used for in cancer?

off-the-shelf cell therapy

Where could IPSCs be used in cancer?

Autologous iPSCs could act as personalized cancer vaccines – treatment possible within a few weeks of diagnosis

What was administered along with the induced pluripotent stem cells (cancer) to stimulate immunity?

An adjuvant.

What does the adjuvant mimic to activate the immune system?

Bacterial and viral nucleic acids.

What does the immune response recognise when iPSCs and adjuvant are injected?

The immune system recognizes antigens on the iPSCs.

What happened when a tumour was later inoculated in the mice?

The immune system recognized the tumour antigens and reduced the tumour size.

What type of immune cells were harvested from treated mice?

T cells

What effect did transferring T cells to tumour-bearing mice have?

The T cells trained the immune system of the recipient to fight the tumour and reduce its size.

What is the potential clinical application of this strategy cancer vaccines?

Patient-specific cancer immunotherapy using vaccines based on iPSC antigens.

Why are the induced pluripotent stem cells irradiated before use?

To prevent them from proliferating.

What determines self or not self?

human leukocyte antigen complex
HLAs are co-dominantly expressed

• Inherited as haplotypes

Immune Compatibility of hPSCs?

thought to be immune privileged due to undifferentiated state
Does not seem to be the case
hPSCs need careful HLA matching – not a universal, off-the-shelf cell type

What is immune shielding between mother and foetus?

foetus is not rejected and expresses/lack of expression of certain markers to protect it from recognition

What gene-editing tool was used to knock down the expression of HLA molecules on the surface of cells?

CRISPR-Cas9

What was the purpose of using CRISPR-Cas9 in this experiment?

To remove genes essential for HLA molecule expression and prevent immune recognition.

What test was done after CRISPR editing?

A test to confirm that HLA molecules were no longer present on the cell surface.

What gene was introduced into the cells via lentiviral transfection?

he gene for CD47 (immunosheilding)

What was the purpose of introducing CD47 into the cells?

To help the cells evade immune detection .

What was done after lentiviral transfection?

Cells expressing CD47 were selected

Did the engineered cells retain pluripotent stem cell properties?

they maintained iPSC characteristics.

What cell types were the engineered iPSCs differentiated into for testing?

SMCs (smooth muscle cells), ECs (endothelial cells), and CMs (cardiomyocytes).

What was observed after injecting these cells into allogeneic, mismatched recipients (CD47)?

The cells survived and evaded immune rejection.

What potential application does this method support CD47?

Universal grafts for transplantation.

What broader vision does this technology enable?

Creation of off-the-shelf banks of pluripotent stem cells for multiple clinical uses.