Pluripotent stem cells

What can regenerative medicine do

Induce stimulation of endogenous repair, human tissue transplantation, cell and gene therapies, engineered tissues and xenotransplantation

Aim of regenerative medicine

Replace, repair or regenerate cells, tissue and organs to restore health

What are stem cells

Unspecialized cells which have the ability to self-renew, produce more stem cells and differentiate into other cell types - embyonic, induced pluripotent and adult/somatic s.c

Totipotent

Cell can differentiate into any other cell with in their organism plus extra embryonic tissues - placenta

most plastic

Pluripotent

Any cell within an dult organism

Multipotent

Specialised U

limited to tissue

Unipotent

one lineage - specialised

Detection

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

How can you determine the expression

Western blotting, RT-PCR, Immunocytochemistry, flow cytometry

How do they enrich cell populations

Centrifugation

MACS

Flow cytometry

Density centrifugation

Cell w.in blood can separate dependent on density

most basic

Density centrifugation method

Plasma at top layer, layer of mononuclear cells then Ficoil and RBCs and granulocytes

Get rid of unwanted cells by using cocktail of a.b (tetrameric complex)

1 a.b recognise cell on ag that we want to get rid off and other rec ag on RBC

big aggregate of RBC around unwanted

Centrifuge - RBC and unwanted = increase density to sit at the bottom

Collect enriched cells

Which layer are stem cells found in

Mononuclear cells

MACS magnetic

Activated cell sorting

antibody rec ag on cell of interest/unwanted cell with magnetic antibody

incubate cell with magnet tagged ab to bind to cells we want

Pass thru column with a magnet field which stick the magnet antibody

flush unwanted cells out

takeaway magnet

flush wanted magnet cells

collect wanted cells

Flow cytometry

Method for detecting specific molecules on and with in cells

can also sort/isolate specific sub populations cells

most powerful

Flow cytometry in medicine

Immunophenotyping

diagnosis

cell sorting

what are the 2 main detectors in flow cytometry

1. FSC, forward scatter cell detector

   1. SSC, side scatter cell detector

How does Flow cytometry work

Cells pass rapidly thru laser

when they hit laser beam they dim the light

FSC = detect dimness of light = longer dimming = bigger cell

SSC = detect side scatter of light from cell = increased ssc = bigger cell

can label cells with multiple antigens

dicromeric mirrors reflect different waves of light - different colours of light to different detectors

up to 6 lasers

FACS, fluorescent activated cell sorting

Flow cytometry but a.b used with fluorescent tag

mainly used for proteins - DNA and RNA can also be detected

can measure up to 18 colours

emerge in droplet of buffer where they ca be given a charge to separate cells

unlabelled aren’t given a charge so pass straight thru

colours then go to oppositely charged electrode

2 types of pluripotent stem cells

1. ESCs

2. iPSC

Embryonic stem cells

Derived from blastocyst at day 5/6

inner cell mass cells derived and then grown in culture and can differentiate

can differentiate into all germ layers

Master regulators of pluripotency - iPSCs

OCT-4

SOX-2

NANOG (transcription factor)

Lin -28 is RNA binding protein rest are TF

also c-MYC and KLF-4

Issues with ESCs

Ethical issues - derived from embryo

teratoma risk - grow indifferently

allogenic - non self

iPSCs, induced PSC

Take adult fibroblasts and expose them/transfected them with genes known to be involved in pluripotency (ESCs)

Similar morphology, unlimited proliferation, surface markers, gene expression, differentiate into cells of all 3 germ layers

Potential for hPSCs

transfect someones cells with pluripotency to reprogramme to produce patient specific pluripotent SC w/o rejection

used to make disease models in la for drug testing, correct genes, infused into patient, regenerative tissue

can differentiate into all cell types in the body incl SC

Stem cell niches

microenvironment in which a cell finds itself

niche regulates cell function

controlled by cell interaction, cell matrix interaction, soluble factors

want to replicate the conditions in particular tissue and signalling for specific differentiation

Directed differentiation

mechanical properties of the growth surface can influence sc fate

change phenotype of a cell by changing elasticity of the surface the cell is growing on

Personalised cancer vaccines

Fibroblasts turned into iPSCs

Autologous iPSCs could act as p.c.v.

iPSCs express a number of tumour associated antigens to train immune system to fight against a tumour

they have been eradicated so they cant proliferate or form a teratoma

Transplant rejection

Donor fibroblasts

HLA recognise cells as a non-self

large combinations of HLAs on surface

if recipient HLA isnt matched it is sseen as non self and risks rejection

careful matching

cant be used as off the shelf cell type due to HLA - class 1 markers on surface when differentiated these levels can change = careful matching

Hypoimmunogenic iPSCs

interface between mother and foetus revealed molecules may be involved in immune shedding n

foetus isnt rejected and expresses markers to protect from recognition as non self - exp CD47 - avoid phagocytosis in tumour cells

Hypoimmunogenic iPSCs advancement

Gene editing - knock down HLA molecules on surface ofcells

reduced exp of HLA 1 and 2 after CRISPR CAS 9

Transfected with lenti virus containg CD47 gene

no immune response or teratomas

used as off the shelf

non recognition

lacked HLA molecules but increase CD47