Lecture 1 - General introduction Flashcards
What is regenerative medicine?
Any treatment that restores normal function to tissues or organs, often using body’s natural healing abilities
What is the aim of regenerative medicine?
To replace, repair or regenerate cells, tissues and organs to restore health
What are the applications of regenerative medicine?
Endogenous repair, cell and gene therapies, engineered tissues, xenotransplantations, human tissue transplantation
What are autologous stem cells?
Cells or tissues obtained from the same indivdual (self-cells)
What are allogenic stem cells?
Tissues or cells which are taken from a donor individual of the same species
Process of obtaining stem cells
- Harvest
- Selection
- Amplification
- Differentiation
- Screening
- Administration
How is directed differentiation stimulated?
ESC/iPSCs converted to cell fate by processes similar to in vivo development
Growth factors and small molecules push differentiation. E.g. Activin A -> Endoderm - FGF10, RA -> Hepatocyte precursor - FGF4, HGF, EGF -> Hepatocyte
Methods of gene therapy
Direct delivery:
- treatment added to vector, e.g. AAV, delivered to pateint
- e.g. cystic fibrosis
Cell-based delivery:
- treatment added to harmless retro/lenti-virus and cultured with autologous stem cells which introduces treatment into isolated stem cells, stem cells containing treatment returned to patient
- e.g. haemopoetic stem cells often used to reprogram blood system
Strimvelis: first ex vivo stem cell gene therapy
- Treats severe combined immunodeficinecy (SCID) caused by adenosine deaminase deficiency
- Autologous CD34+ cells expressing functional ADA
Gene therapy methods
- Elimination (or insertion) of mutation
- Replacement of faulty gene
- Insertion of therapeutic gene
- Transcriptional regulation
What are ex vivo gene editing methods?
- T-cells
- HSCs
- iPSCs
What is xenotransplantation?
The transplantation of cells, tissues, or organs between different species
What are the benefits of xenotransplantation?
Potential solution to shortage of human organs for transplantation
Example of xenotransplantation
Moazami et al (2023) performed two heart xenograft transplants of porcine into two brain-dead human recipients. Both xenografts had excellent cardiac funcion immediately after transplantation, one had cardiac deceline due to size mismatch between donor and recipient. Also performed successfully without hyperacture rejection or zoonosis.
How are stem cells identified?
Identified by expression or absence of a number of transcription factors and cell surface markers
What are the applications of stem cell markers?
Used to enrich stem cell populations for further study of biomedical applications, e.g. tissue engineering
Methods to determine expression of stem cell markers
- WB
- RT-PCR
- Immunocytochemistry
- Flow cytometry
Methods to enrich stem cell populations
- Crosslinking and centrifugation
- MACS
- Flow cytometry/FACS
Method of density centrifugation
- Density gradient medium, e.g. Ficoll-Paque prepared
- Sample layered on gradient
- Sample is centrifuged and stem cells typically appear in mononuclear cell layer
- RosetteSep antibody cocktail selectively removed unwanted cells through antibody-mediated immunorosette formation (unwanted cells crosslinked to RBCs forming dense pellets)
- Leaves enriched stem cells at interface between plasma and the density gradient medium
Method of Magnetic-Activation Cell Sorting (MACS), and different selection stratergies
Magnetic beads isolate specific cell types or subpopulations from a mixture.
- Magnetic beads coated with antibodies that bind specific surface marker on cell of interest
- Mixture passed through magentic separation column and beads retained in colum by magentic field
Magentic field removed and retained cells are eluted from column
Selection strategies:
- Positive selection
- Untouched isolated (bind unwanted cells)
- Sequential sorting (untouched isolation followed by positive selection)
Method of flow cytometry
Detects specific molecules on and within cells - sorts and isolates specific subpopulations of cells.
Basic:
- Cells suspensed in fluid creating single-cell suspension
- Sample passed through flow cytometer which interacts with laser beam
- Cells scatter the laster light, the intensity and angle of scatteredd light indicative of cell size and granularity
Extras:
- Fluorescently labelled antibodies or probes added to suspension to bind specific markers on cell surface or within cell
- Multiple antigens can be quantified simultaneously
- Mainly used for proteins, but DNA and RNA can also be detected
Method of Fluorescent-Activated Cell Sorting (FACS)
Isolates specific cell popultions based on their fluorescent characteristics. Same as flow cytometry but with fluorescene used to sort. Flow cytometer applies an electrical charge to cells of interest that directs charged dripletes into separate collection wells.
Example of FACS
- Flow cytometry to detect STRO-1 shows 3 populations of STRO-1+ cells
- 3 different populations identified by gating graphs
Enriches MSCs
