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

most plastic

Pluripotent

Any cell within an dult organism

Multipotent

Specialised U

Unipotent

one lineage

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

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 NP

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

Pass thru column with a magnet

flush cells out

collect wanted cells

Flow cytometry

Method for detecting specific molecules on and with in cells

can also sort/isolate specific sub populations cells

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

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

2 types of pluripotent stem cells

1. ESCs

2. iPSC

Embryonic stem cells

Derived from blastocyst at day 5/6

inner cell mast cel89ls derived and then grown in culture and can differentiate

Master regulators of pluripotency

OCT-4

SOX-2

NANOG (transcription factor)

Issues with ESCs

Ethical issues

teratoma risk

allogenic

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 is recruited as a non-self

large combinations

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 when differentiated these levels can change = careful matching

Hypoimmunogenic iPSCs

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

non recognition

lacked HLA molecules but increase CD47