Mesenchymal Stem Cells

Where is bone marrow typically obtained from?

Iliac crest

How can you culture MSCs?

Obtain bone marrow, density centrifugation (mononuclear cell layer) and plate, HSCs don’t adhere to plastic and will get removed over time (lag phase) to leave a confluent layer of fibroblast-like cells that exponentially grows.

What is the morphology of MSCs in culture?

Adherent, spindle-shaped, fibroblastic colonies in culture

What do MSCs differentiate into?

Adipoctyes, chondrocytes, osteoblasts

What media supplements are needed for adipogenesis (4)?

IBMX, dexamethasone, insulin, indomethacin

What media supplements are needed for chondrogenesis (4)

TGF-B3, serum free, in high density to aggregate (3D pellet culture) and under hypoxic conditions.

How can chondrogenesis be enhanced?

IGF-1, BMPs 2,4,6,12,13.

What media supplements are needed for osteogenesis (4)?

Dexamethasone, B-glycerol phosphate, ascorbate, 10% FBS

How can osteogenesis be enhanced?

BMP2, bFGF

What is the common stain for adipocytes?

Oil red O (fat soluble dye in the neutral lipid environment of fat cells)

Why is the common stain for chondrocytes?

Collagen II (main ECM molecule in cartilage) and toludine blue (stains glycosaminoglycans found in ECM of cartilage tissue)

What is the common stain for osteoblasts?

Alkaline phosphate and alzarin red (stain calcified matrix/bone nodules)

What is the master transcription factor for osteoblasts?

RUNX2 (inhibited by PPARY)

What is the master transcription factor for adipocytes?

PPARY (inhibited by wnt)

What is the master transcription factor for chondrocytes?

Sox9 (inhibited by RUNX2, wnt, PPARY)

Explain commitment to chondrocyte lineage.

Sox9 upregulated which causes MSC condensation/aggregation. Positive feedback loops including BMP promote further sox9 expression.

Explain the pre-hypertrophic chondrocyte stage.

Cells express collagen II and aggrecan (hyaline cartilage) and Indian hedgehog and collagen X and secrete MMPs

What is hypertrophy?

Cells increase in size, metabolic activity and volume.

Explain the hypertrophic stage.

Cells increase in size, accumulate calcium and reduce collagen II production. Wnt promotes sox9 expression. Positive negative feedback occurs depending on where cells are in cartilage tissue.

Explain the positive negative feedback loop.

PTHrP delay’s chondrogenesis during cartilage development to prevent premature hypertrophy in the perichondrium by inhibiting Indian hedgehog.

How does TGF-B3 enhance chondrocyte differentiation?

It activates SMAD3, which activates Sox9 and inhibits RUNX2.

What are the minor lineages of MSCs?

Muscle myoblasts, stromal cells, tendon tenocytes.

Why is there no definitive marker for MSCs?

It’s a very heterogenous population that are not functionally equivalent and are found everywhere around the body.

What can be used to define MSCs?

Adhere to plastic in culture, express CD105, CD73, CD90, lack expression of CD34, CD45, CD14 or CD11b, CD79a or CD19, HLA-DR, and differentiate into bone, cartilage and fat.

What are some of the differences in function between MSCs?

Differences in surface markers, proliferation and differentiation potential (bone marrow, vena cava and muscle MSCs have differences in differentiation and proliferation potential).

What is the best source of MSCs for therapeutic use?

Adipose because it has the highest number of cells isolated (557±673) compared to umbilical cord that is limited, and is less painful that bone marrow.

Explain the heterogeneity of MSCs.

Tissue origin (niche), developmental stage (some are more primitive and tripotemt where others may be more committed and unipotent), epigenetic landscape, cellular ageing (replicative senescence with lower differentiation potential and different markers like CD44 increase/CD90 decrease).

Why is cord blood popular for storage?

MSCs have the highest ability to differentiate and replicate so could be a ready source for later life.

What is the central role of MSCs?

Tissue homeostasis (repair, regeneration, secretion of paracrine factors recruit cells and immunomodulate, reduced ability with ageing).

What 3 interactions influence the MSC niche?

Physical/mechanical factors with ECM, soluble signals, cell-cell interactions

Where is the MSC niche?

In the bone marrow. Basement membrane of the perivascular niche (surrounding blood vessels especially sinusoids) or the Endosteal niche (inner surface of bone).

Why is it important they exist in the basement membrane?

So they can be induced to circulate in the blood and differentiate into cells that aid in repair in response to disease/damage OR can migrate to other tissues.

How does substrate stiffness (mechanical properties) affect the differentiation potential of MSCs?

Stiffness affects signalling through the cytoskeleton to nucleus which affects transcription. Brain related genes (GDNF) are upregulated on soft substrates, myogenic genes (MYOG) on moderate, and osteogenic genes (BGLAP) on stiff. Morphology also differs. Soft (neurite-like processes), moderate (spindly fibroblastic), stiff (spread out and flat).

Why could the role of MSCs be argued?

Mechanisms have been studied in vitro so taken out of their niche

What is the paracrine function of MSCs?

MSCs can educate (modulate and instruct) other cells through paracrine signalling, cell-cell interactions and extracellular vesicles (transport proteins and nucleic acids - selective packaging and exclusion).

What is the use of MSC EVs (MSC secretome)

Cell-free therapies to deliver mRNA for translation of cytokines/miRNAs/growth factors/neurotrophic factors more efficiently than exogenous application.

What is the immunogenicity of MSCs?

Generally regarded as hypoimmunogenic (not immune-privileged) as they have low MHC expression and secrete immunomodulatory factors.

What are the therapeutic uses of MSCs?

Treatment of GvHD to improve transplant outcomes, therapy following stroke/heart attack because they home to distant site of injury, delivery of therapeutic proteins, MSC secretome education, tissue engineering to make bone, cartilage and fat tissue.

What is MSC priming?

Exposing MSCs to specific stimuli before therapeutic use to change their properties like migration/homing, immunomodulatory, regeneration.

What are the 5 common priming methods?

Cytokine, hypoxia, TLR, pharmacological, 3D culture/mechanical.

How MSCs be used to treat myocardial infarction?

Paracrine signalling secrete a range of factors to stimulate angiogenesis/anti-apoptosis/anti-fibrosis/pro-survival/migration (VEGF, HGR, IGF-1, SDF-1, TGF-b). Immunomodulation to dampen post farction inflammation and support functional preservation. Stimulate endogenous cardiac repair by stimulating resident CPCs/CSCs (cardiomyogenesis and vasculogenesis).

Why do MSCs home to cancerous tissue?

Cancers are sites of injury that never heal. Inflammatory cytokines mobilise MSCs and attract them to cancer site (like immune cells) in attempt to repair.

What guides MSCs to tumours?

Cytokines and chemokines.

How is the TME involved in MSC behaviour?

Signalling is very complex and influenced by hypoxia, ECM composition, extracellular acidity and inflammatory stromal components.

Do MSCs favour tumourogenesis or anti-tumour growth?

The factors secreted by MSCs are stacked in favour of tumourogenesis.

What factors are pro-tumourogenesis?

CCL5, TGF-B, IL-6, IL-10, VEGF, NEUROREGLIN, MMP, SDF-1

What factors are anti-tumour growth?

Oncostatin-M

What happens when MSCs are primed by TLR4?

Pro-inflammatory phenotype that is anti-tumour.

What happens when MSCs are primed by TLR3?

Anti-inflammatory phenotype that promotes tumour growth and metastasis (good for inflammatory/autoimmune diseases).

How can MSCs be genetically modified for cancer therapy?

Can be genetically engineered like a trojan horse to produce therapeutic proteins (from a transgene) such as TRAIL that induces apoptosis in cancer cells and home to tumour injury sites for targeted delivery.

What is the function of TRAIL in cancer therapy?

TNF-related apoptosis-inducing ligand promotes apoptosis specifically in cancer cells (differential expression of death receptors on cancer cells vs healthy cells).

How are MSCs used in photodynamic cancer therapy?

MSCs are loaded with a photosensitiser that produces free radicals when exposed to light, leading to targeted disruption of cancer cells.

What is the Trojan horse strategy in MSC-based cancer therapy?

It involves engineering MSCs to carry therapeutic agents (e.g., photsensitisers, proteins)directly to tumours, exploiting their natural tumour-homing ability.

How can MSCs be used as drug carriers in cancer treatment?

MSCs can be loaded with chemotherapeutic agents or nanocarriers (TRAIL, DOX, endostatin, miR-124) which they transport directly to the tumour site for localised drug release and reduce systemic toxicity.