lecture 4 Flashcards
(12 cards)
How are ES cells formed?
Fertilisation leads to blastocyst formation – inner cell mass contains pluripotent stem cells which are isolated and put onto feeder layer
Induce differentiation
ES cells do not yet have huge future in regenerative medicine due to risk of teratoma formation, need to be allogeneic
Could be used for supply of secreted products but currently not as cell therapies
What are iPS cells?
Adult human cells transduced with small sets of genes (4 or fewer) that reprogramme the adult cells to give embryonic stem cell characteristics.
Large role in regenerative therapies
Can differentiate, pass teratoma assay
Can induce reprogramming and differentiation
Opportunities…
…and challenges
Reprogramming is very inefficient, often less than 1% of the cells that incorporate the reprogramming factors revert to iPS cells.
Use of viruses.
Risk of uncontrolled cell growth.
Epigenetic memory.
Opportunities…
…and challenges
Reprogramming is very inefficient, often less than 1% of the cells that incorporate the reprogramming factors revert to iPS cells.
Use of viruses.
Risk of uncontrolled cell growth.
Epigenetic memory.
What are adult stem cells?
Undifferentiated cells found in a variety of different adult tissues.
ASCs can be isolated, expanded and induced to differentiate into different lineages.
Examples of Adult Stem Cells
Haematopoietic stem cells (Stage 1 & 2)
Epidermal stem cells (Stage 1 & 2)
Neural stem cells (Stage 2)
Limbal stem cells (Stage 2)
Mesenchymal stem cells (Stage 2)
What are mesenchymal stem cells?
Most widely used cell type for clinical therapies.
Therapeutic effects believed to be mediated predominantly by:
1) Differentiation into new tissues.
2) Secreted factors, the so-called “paracrine effect”
MSCs can serve a number of different functions including differentiation to skeletal lineages, tissue organisation, haematopoietic support, immune regulation (often immune suppression) and vascular organisation.
What is MSC heterogeneity?
Significant variation exists in MSC cultures isolated from different donors and different tissue sites. Unrefined and non-standardized isolation and culture techniques do not select for homogeneous cell populations and are likely to give rise to a mixture of stromal cell with different functions. Differences in the growth properties of MSC clones can result in cultures being dominated by the faster-growing lines. Further levels of heterogeneity can be introduced within MSC clones through asymmetric cell division and the effects of stochastic transcriptional noise, generating cells with modified phenotypes. MSC properties will also be determined by, for example, proximity to neighboring cells and extrinsic signaling factors.
Transcriptional noise- variation in gene expression profiles
Location in colony determines growth signals received
What are the functions of extracellular vesicles?
EVs carry a complex cargo of signalling molecules capable of influencing cells
Carry microRNAs, DNA, mRNA
Have characteristic cell surface markers
Therapeutic potential:
EV appear to replicate the therapeutic benefits of the parent cell (e.g. iPSCs, MSCs).
May be use for delivery of native bioactive molecules (e.g. miRNAs, proteins) for repair and regeneration, modulation of immune responses.
Additionally, as carriers for delivery of small molecules, pharmaceuticals, siRNAs, CRISPR/Cas9.
EVs are formed by two mechanisms. Exosomes are formed by the endocytic pathway through invagination of the endosomal membrane, which forms multivesicular bodies (MVBs) that can fuse with the plasma membrane to release exosomes into the extracellular milieu. Microvesicles (MVs) arise from the outward budding and fission of the plasma membrane. All subtypes of EVs share a general composition of an outer lipid bilayer and various proteins, lipids, and nucleic acids carried by the vesicles. The specific content of EVs is largely dependent on biogenesis, cell source, and culture conditions. EVs have been suggested to be internalized into target cells by various uptake mechanisms including membrane fusion (171) and different endocytic pathways including phagocytosis (172), receptor-mediated endocytosis (173), lipid raft–mediated endocytosis (174), clathrin-mediated endocytosis (175), caveolin-mediated endocytosis (176), and macropinocytosis (176).
Evade immune responses so have advantages over traditional carrier systems
Explain why MSCs Are a Particularly Attractive Cellular Platform for Cell-Based Cardiac Regenerative Medicine
MSCs can be isolated from virtually all tissues, including bone marrow, adipose tissue, umbilical cord, and the heart itself, or they can be generated from pluripotent stem cells. Ongoing research, examining the relationship of MSCs to heart development and repair, will guide the development of more refined strategies, such as isolating bona fide cardiac MSC lineages, generating genetically engineered MSCs with better defined growth factor and exosome secretomes, and optimizing combination strategies of MSCs with cardiac progenitor cells and/or biomaterials more relevant to cardiac organogenesis. Translation of this research should address the most important clinical concerns, including feasibility, safety, and efficacy of utilizing MSCs as the basis for cell-based and novel cell-free, autologous and/or allogeneic regenerative medicine approaches. These approaches are designed to safely and effectively augment MSC repair of the damaged heart by delivering microvesicles/exosomes and growth factors and by cell-cell coupling that together activate anti-inflammatory and antifibrotic pathways and stimulate the proliferation of endogenous cardiac precursors, cardiomyocytes, and coronary vascular cells.
What is cell engineering?
The process of modifying cells for therapeutic applications.
Can involve mechanical engineering, reprogramming (iPSC technology), genetic modification, genome editing (CRISPR/Cas9).
What are genetic engineering and gene therapy?
The genetic manipulation of a cells for therapeutic purposes.
Over-expressing a gene with therapeutic value
Introducing a new gene
Deleting or inactivating a mutated gene
Replacing a mutated gene with a normal gene
Increasing focus on genome editing using CRISPR/Cas9.
DNA introduced to cells by non-viral (e.g. lipophilic carriers, electroporation, sonification, gene gun) and viral delivery .
What is CRISPR/CAS9 Genome Editing?
CRISPR-Cas9 gene editing technology can be used to precisely disrupt and modify specific genes.
Guide RNA designed to recognise target sequence connected to Cas9, taking Cas9 through that target sequence and cleavage of the double strand
The cell can either repair that erroneously to create a deletion in that area and have a loss of function due to NHEJ
Or if you provide a template you can introduce a new sequence through HDR
Flexible way of editing genome by deleting/inserting genes
What are suicide genes?
Encode non-toxic proteins in genetically modified cells, which when exposed to a specific prodrug, becomes toxic to the cell and kills it.
Most common suicide gene is herpes simplex virus thymidine kinase (HSVtk). When exposed to ganciclovir (GCV, the prodrug), it initiates the conversion by endogenous kinases to GCV-triphosphate.
HSVtk converts GCV to monophosphate form.
GCV-triphosphate induces apoptosis by inhibiting DNA replication
if worried about teratoma, insert suicide gene to kill that population
Fail safe way to control population
Conversion- series of phosphorylation events that trigger apoptosis
If suicide gene carried within donated cells exposure to prodrug will kill off the cells
What are gene activated matrices?
GAMs = Genes, siRNAs, CRISPR/Cas9 packed into viral or non-viral delivery systems within a matrix
Provides sustained local delivery
matrix could be collagen, synthetic polymer etc to deliver a gene
