Wnt Signaling and Beta-Catenin Regulation

Wnt Signaling

• Canonical WNT signaling occurs when no Wnt ligand is present, Frizzled is not activated.
• A destruction complex of Axin, APC, CKIα, and GSK-3β phosphorylates β-catenin, leading to its ubiquitination and degradation, preventing transcription.
• When Wnt is present and binds to Frizzled, the destruction complex is inactivated, stabilizing β-catenin.
• Stabilized β-catenin accumulates and travels to the nucleus, binding TCF/LEF1 to transcribe target genes.
• Wnt signaling is vital for cancer, embryonic development, and adult stem cell maintenance.
• High Wnt levels promote stem cell maintenance, while Wnt removal leads to cell differentiation.

GSK3 or AXIN KO

• Knockout (KO) of GSK3 or AXIN, components of the β-catenin destruction complex, prevents β-catenin breakdown.
• Prevents breakdown of beta catenin so Wnt pathway is always active, independent of Wnt ligand presence.
• Results in cystic intestinal organoids due to the continuous activation of the Wnt pathway, inhibiting differentiation.

RNF43/ZNRF3

• These are transmembrane ubiquitin ligases that ubiquitinate the Frizzled receptor, leading to its internalization.
• Internalization of Frizzled receptors reduces the potency of the Wnt cascade, downregulating signaling.
• Double knockout (KO) of RNF43/ZNRF3 results in more Wnt activation due to increased Frizzled receptors on the membrane.
• More Frizzled receptors on the membrane leads to cystic intestinal organoids
• Wnt signaling is still needed, RNF43/ZNRF3 are just regulators

Lgr5

• Intestinal stem cell marker able to counter the effects of RNF43 and ZNRF3, by binding to R-spondin.
• The R-spondin ligand binds to LGR5 on the cell membrane, sequestering RNF43 and ZNRF3.
• Sequestration of RNF43/ZNRF3 causes more receptors available for Wnt signaling.

Lineage Tracing (Crypt)

• LGR5 marks crypt base columnar cells (CBCs) that act as stem cells.
• These cells can be visualized by inserting GFP behind LGR5 in the DNA, causing green fluorescence only in the crypt base.
• Inserting a Cre gene after LGR5 allows Cre enzyme expression in LGR5+ cells.
• Cre enzyme can remove stop codons in front of a LacZ region, leading to blue color expression in the cell and its progeny.
• LacZ expression is retained even after cell differentiation, allowing visualization of the descendants of LGR5+ cells.
• This method is called lineage tracing.

Noggin

• BMP inhibitor is used to inhibit differentiation.

EGF

• Growth factor, used for intestinal cell proliferation.

ENR Medium

• ENR medium contains:
  • EGF which promotes intestinal cell proliferation.
  • Noggin which inhibits BMP to prevent differentiation.
  • R-spondin acts as a Wnt agonist to maintain stem cell properties.
• Matrigel (extracellular matrix) forms a hydrogel supporting structure with collagens and laminins.
• This combination forms gut-like structures known as organoids.

R-spondin

• Wnt agonist that promotes stem cell maintenance.
• It can bind to LGR5 on stem cells, which sequesters RNF43 and ZNRF3.
• Sequestration leads to more available receptors for Wnt signaling.

Intestinal Homeostasis

• The intestine is composed of crypt and villus regions.
• Crypts contain stem cells and Paneth cells.
• Paneth cells produce cells that migrate up the crypt and begin differentiation.
• Cell exclusion occurs at the top of the villus within 5 days.
• Wnt signaling is high in the crypt, maintaining stem cell properties.
• Wnt is produced by Paneth cells (also EGF and DLL) and the mesenchyme (fibroblasts and myofibroblasts).
• Wnt target genes include LGR5 and RNF43/ZNRF3.
• LGR5 binds R-spondin, inhibiting RNF43/ZNRF3.
• RNF43 and ZNRF3 inhibit Wnt signaling by stimulating ubiquitination.
• EGF promotes intestinal proliferation.
• Noggin inhibits BMP, expanding crypt numbers.
• R-spondin is a Wnt agonist for stem cell maintenance.

Organoids

• Three-dimensional in vitro cultures grown from stem or progenitor cells.
• Cells self-organize through cell sorting and spatially restricted cell lineages.
• Organoids can be formed from pluripotent or adult stem cells of a tissue.

Applications of Organoids

• Biological applications include:
  • Studying basic gene functions like WNT signaling.
  • Investigating cellular and molecular processes.
  • Studying signaling pathways.
• Translational applications include:
  • Studying pathogen-host interactions in infectious diseases.
  • In vitro disease modeling (hereditary diseases, cancer).
  • Toxicology studies.
  • Personalized medicine by using adult stem cells from healthy individuals or generating mutations to create tumor-like organoids.
  • Drug testing and screening.
  • Regenerative therapy via organoid transplantation.

Function of Kidney

• Kidneys regulate homeostasis.
• They are involved in hormone secretion/regulation.
• Kidneys perform nutrient reabsorption and excretion.
• Homeostasis functions include:
  • Secretion and reabsorption of molecules and ions (Na+, K+, Ca2+, Mg2+).
  • Acid-base balance (H+, HCO3-).
  • Nitrogen waste product removal (urea, uric acid).
  • Water regulation.

APC Mutation

• APC mutation leads to Wnt upregulation, which can cause cancer.
• The ability to undergo metastasis results from loss of dependency on niche-specific signals, such as after APC knockout.
• Other cancer mutations include TP53, SMAD4, and KRAS/BRAF.

KRAS/BRAF Mutation

• EGF is upregulated and BMP is downregulated
• Cancer mutation.
• Other cancer mutations include TP53, SMAD4, and APC.

Tumoroids

• Organoids can be made from normal or tumor tissue to resemble a patient's organ.
• Tumoroids can be used for drug screening before starting therapy.
• Screening can help predict the clinical outcome and prevent unnecessary treatment.

Kidney

• The kidney consists of an outer renal cortex and an inner renal medulla.
• The functional units of the kidney are nephrons.
• Nephrons consist of the glomerulus and tubules leading to the ureter.
• Glomeruli, located in the cortex, are essential for blood filtration.
• The tubular parts have homeostatic and endocrine functions:
  • Selective secretion and absorption.
  • Production of erythropoietin and renin.

Regenerative Capacity of Kidney

• The kidney has limited regenerative capacity and cannot regenerate lost nephrons.
• Nephrogenesis cannot occur after birth.
• Troy+ cells are kidney stem cells, present during embryonic development, but minimal postnatally and in adults.
• Treatments for kidney failure are dialysis or transplantation.

Chronic Kidney Disease

• 10% of the population has chronic kidney disease (CKD), especially the elderly.
• Many people are unaware they have it.
• The number of cases is increasing.
• In the Netherlands, 10,000 people have transplants, and 6,500 are on dialysis.
• Dialysis has a survival rate of 5 years.
• CKD accounts for 2% of healthcare spending.
• The final outcome of kidney disease is end-stage kidney disease (ESKD), with less than 10% kidney function remaining.

Current Therapeutic Approaches for ESKD

• Dialysis removes waste and excess fluid from the blood.
  • Advantages: Relatively simple, rapid exchange of small molecules.
  • Disadvantages: Does not replace all kidney functions, intermittent, heavy impact on patient's life, >20% mortality/year.
• Transplantation replaces all kidney functions.
  • Advantage: True renal replacement.
  • Disadvantages: Shortage of donor organs, rejection, maximum 10-year lifespan, immunosuppression leading to infection and malignancies.

Bioartificial Kidney vs. Bioengineering of a Kidney

• A bioengineered kidney aims to introduce a generated organ or functional tissue, mimicking transplantation.
• A bioartificial kidney mimics dialysis by only taking over part of the kidney functions.

Kidney Dialysis: Pros and Cons

• Hemodialysis and Peritoneal dialysis
• Pros: simple, fast
• Cons: Not all kidney functions are replaced (no exchange of middle and large molecules, no active transport, no hormone production, no neurological regulation), intermittent, impact patients life, 20% mortality/yr.

Bio-artificial Kidney

• Normal kidney dialysis does not allow passage of larger molecules, which is a problem for protein-toxin bound molecules.
• Requirements:
  • Membrane:
    • Cell membrane adhesion.
    • Clearance on fiber.
    • Scalability in vivo biocompatibility.
  • Proximal tube cells with transporters:
    • Controlled growth.
    • Low immunogenic response.
    • Proper transporter function.
    • Not tumorigenic.

Development of Cell Line for Bio-artificial Kidney

• Process of developing a cell line:
  • Immortalization of cells.
  • Using cells from urine and kidneys.
  • Controlled growth by integrating an oncogene that proliferates only at a certain temperature.
  • OAT1 is an essential transporter examined using fluorescence to track uptake.
  • Immune activation is checked.
  • Oncogenicity potential is checked by injecting cells into mice and looking for tumor formation.

Developing Adherence Material for Bio-artificial Kidney

• Use of living membranes.
• Muscles can bind to any surface with L-dopa, which uses hydrogen bonds, covalent bonds, and metal bonds.
• Collagen type 4 (IV) is used.
• Cells adhere with muscle and collagen IV (and PES HF).

Developing Active Transport Across Membrane for Bio-artificial Kidney

• Kidney on a chip to research transport over membrane.
• Tracked fluorescent compounds for specific transporters, indicating uptake.
• Protein-bound toxins were also decreased using this.
• Control: Blocked transporter resulted in only unspecific uptake and not removal of protein-bound toxins.
• Experiment comparison with protein-bound toxins vs. removal of loose toxins:
  • Bound form (indoxyl sulfate) had more transport.
  • Suggests protein facilitates transport.
• Albumin in kidney patients is different and toxins bind even stronger, which results in less clearance.

Challenges in Bio-Artificial Kidney

• Scalability: Up to 5 fibers currently, needing to increase surface area.
• Packing of fibers requires inner seeding:
  • Ensuring proper monolayer.
  • Minimizing fiber size for better adhesion.
  • Coiling fibers using bioprinting to make perfusable models.
• Alternatives to fibers:
  • Using silica wafers to improve filtration due to defined pores but this make them more fragile and have limited porosity.
• Demonstration of in vivo safety and efficacy.
• Moving from extracorporeal to implantable systems for less clinic visits.
• Expanding tubules for drug nephrotoxicity screenings like liver and gut on chips.

Organoids and Stem Cells for Bioengineered Kidney iPSC

• Generating kidney organoids from human iPSC in vitro:
  • Using Yamanaka factors for resetting, then exposing cells to CHIR and FGF9. Organoid pros:
  • Organized near-physiological nephron structure with glomerulus and tubule.
  • Contain epithelial and mesenchyme cells. Organoid cons:
  • Cells stay immature; lack transporters.
  • Teratoma formation risk.
  • Inefficient reprogramming.
  • Lack vascularization and urine collection system.
  • Not clinically compatible and time-consuming.

Organoids and Stem Cells for Bioengineered Kidney ASC

• Generating kidney organoids from adult stem cells (ASC) = tuboloid (from kidney tubular epithelial cells)
  • Using urine or kidney biopsy to dedifferentiate to progenitor cells which resemble adult stem cells. Organoid Characteristics:
  • Cystic morphology (mimics tubules).
  • Single layered cuboidal epithelium.
  • Genomic stability.
  • Polarization.
  • Leak-tight.
  • Selective transport.
  • Easily derived. Organoid pros:
  • Easy to expand, stable, autologous, leak-tight. Organoid cons:
  • No glomerulus,
  • No mesenchyme,
  • Not clinically compatible (animal derived matrigel etc.).
  • No vascularization.
  • No common urine collection system.

Leak-Tight Assay

• Used to testing for kidney organoids
• Inulin-FITC: An increase in fluorescence intensity indicates leakage.
• TEER: High electrical resistance indicates a nice monolayer.

Selective Transport Assay

• Used to testing for kidney organoids
• CDFDA is placed in a basin and with a cell layer, where fluorescence is then checked.
• A control uses a transporter inhibitor.

Challenges with Bioengineered Kidney

• Challenges with bioengineered kidneys:
  • Differentiation
  • Proliferation
  • Scale and size
  • Matrigel/ECM
  • Structure/organization
  • Integration
  • Function

Applications of Kidney Organoid Culture

• Applications include:
  • In vitro modeling of nephrogenesis
  • Ex vivo studies of organ (patho)physiology
  • Disease modeling
  • Drug and toxicity screening for personalized medicine
  • Bioartificial kidney development
  • Bioengineering of the kidney

Bioengineered Kidney Use: 4 Examples

• Bioengineering whole kidneys: Scaffold seeding with decellularized whole kidneys.
• Transplantation of PSC kidney organoids: Using a sheet of iPSC as a "band-aid" on the kidney.
• Bioprinting kidneys (or one nephron): Printing cells and hydrogels.
• nKSPC therapy in the kidney: Administration of nKSPC induces de novo SIX2 activation in proximal tubules

Liver Histology

• Blood enters the liver via the portal vein (80%), derived from the intestines.
• Autologous transplantation: Cell isolation, expansion, co-culture with support cells, spheroid quality control, extrusion-based bioprinting, and transplantation is performed.
• Replacement of animal models is done to show toxicity better via toxicity tests with bioprinting liver organoids and mimicking blood flow; focus on personalized medicine
• Difficulties: dedifferentiation happens fast, need for vascularization in spheroids, fusing to recipient tissue.

Spheroid Formation and Use

• Liver organoids used in 3D via dual extrusion nozzle: hydrogel = MMP degradable, PEG based, hydrolytically cleavable and vasculature with endothelial cells and gelatin based.
• Spheroid formation: Gravity-based triculture (liver organiods ICOs, mesenchymal source, HUBECs) 120 micrometers within 4 days.
• Transplanting spheroids:
• Personalized medicine and testing drug toxicity.
• Whole organ engineering.
• Transplantation?
• Spheroids automatically fuse with tissue. Vasculature tests with immune deficient mice with a with dye to show connectivity and proved that the vasculature in the construct should connect to the vasculature

Vascular Tissue Engineering (VTE)

• Vascular Tissue Engineering (VTE) assembles functional constructs to restore, maintain, or improve damaged tissue or whole organs by creating new blood vessels
• Making new blood vessels within other tissues/organs = vascularized tissue engineering.
• Obstacles:
  • The diffusion limit of oxygen is 200 μm.
  • The limit of host vessel growth is tens of microns/day.

Functions of Blood Vessels

• Primary functions:
  • Enable gas exchange, supply nutrients, and remove waste.
• Other functions:
  • Endocrine signaling, inflammation, anti-coagulation, vasodilation/vasoconstriction, and immunological surveillance.
• Regenerative vascular replacements should perform these functions.
  • Oxygen diffusion limit: 200 micrometers; beyond this distance causes hypoxia.
  • Host vessel in-growth range limit: tens of microns/day.
• All tissues need vascularization.

Need for Vascular Replacement (3 Examples)

• Clinical need for small diameter vessels (≤6mm)
  • Coronary artery disease leads to myocardial infarction (managed with grafts).
  • Chronic kidney disease needs vascular access for hemodialysis (often uses suboptimal Teflon/plastic grafts).
  • Other needs: deep vein thrombosis, artery disease, strokes.

Anatomical Structure of Blood Vessels

• Endothelial lining (HUVECs):
  • Proliferates quickly, selective barrier, anti-coagulant surface, helps vasodilation/constriction, and is a mechanosensor in angiogenesis
• Mural cells (support cells):
  • (Smooth muscle cells and pericytes).
  • Provide structure/stability, manage blood pressure, vascular remodeling, vascular wall pathologies, and ECM production
• Smaller diameter = decrease in blood pressure.
• Diameters:
• Aorta = 25mm
• Femoral artery = 10 mm
• Coronary artery = 3 mm
• Small artery = 100 micrometer
• Arteriole = 5-100 micrometer
• Capillary = 3-6 micrometer
• Below 6 = small artery considered one to be made within vascular replacements

Cardiovascular System Development

• Formation of blood vessels, blood, and heart.
• 1st functional organ system to develop during vertebrate development.
• Vasculogenesis = formation of a capillary plexus without a pre-existing vasculature structure driven by growth of fetus.
• Sequence:
  • Zygote -> blastula -> gastrula -> mesoderm formation -> blood island differentiation -> fusion of blood islands and endothelial cell differentiation -> primary capillary plexus.
• Mesoderm forms circulatory system in 2 different routes:
  • Extraembryonic one: angioblasts + heamapoetic progenitors (blood islands)
  • Intraembryonic one: angioblasts only
• Cells distinguish between artery and vein.

3 Types of Capillaries

• Continuous capillary:
  • Cells are closely bound to each other.
  • Heavily regulated transport via transcytosis.
  • Examples: blood-brain barrier in the brain and blood-air barrier in the lungs.
• Fenestrated capillary:
  • Fenestrae (pores) with diaphragm.
  • Diffusion of fluid and small molecules based on pressure.
  • Example: ultrafiltration in the kidney.
• Discontinuous capillary:
  • Large intercellular gaps and a discontinuous basement membrane.
  • Free exchange of small and large molecules.
  • Examples: liver and bone marrow.

Endothelial Cells

• Cells form the lining of blood vessels and a monolayer with tight junctions, which is a selective barrier between the blood and tissue and anti-coagulant surface
• Play a role in vasodilation, vasoconstriction, mechanosensory, angiogenesis and vasculogenesis
• HUVECs easy to use in the lab and proliferate quickly

Mural Cells

• Mural cells, support vascular system with smooth muscle cells and pericytes, which provides structural stability and vasodilation/vasoconstriction
• Contribute to vasodilation and vasoconstriction, play a role in blood pressure, involved in vascular remodeling and ECM responsible for production in blood vessels

Angiogenesis

• Angiogenesis= capillary sprouting from pre-existing vasculature which is triggered by hypoxia

• Remodeling of primitive plexus to a capillary bed with:

  • High oxygen = low HIF. Low oxygen = high HIF -> VEGF -> formation of tip cells navigate though ECM towards angiogenixc stimulus (VEGFD) and Tip cells will be stalk cells -> from blood vessel.

• Maturation and remodeling by Pruning, remodeling, stabilisation by SMCs:

  • Driven by shear stress and Receptor/ligand driven

• Mechanisms that constirbute to vascular remodeling by Fusion, Introsusception, Sprouting, and Regression

Vascular Progenitor Cells

• 4 types vascular stem cells with varying success of isolation, lack of unique markers, influence of pre-existing disease in fucntiong?

Challenges for use of stem cells for the regeneration of vasculature:

• Lack of unique markers making isolation difficult and vary success of culturing & influence of pre-existing disease on functioning? autologous possible in sick people

Making Regenerative Vascular Replacement

• Engineer a blood vessel on its own and put blood vessels into an organ or tissue. -3 main approaches for vascular engineering:
  • In vitro paradigm using bioreactor culturing and variation on maturation: most laboursome , but best clinically.
  • Autologous graft harversting using the in vivo method: dececularized structure to implant + cells . - In situ TE uses the regenerative potential of the body : use scaffold and directly implant= least laboursome + scaffold breakdown

Paracrine Hypothesis

• Stem cells secrete factors to damaged tissue: secrete facters and anti-inflammatory cytokines to help
• Cells themselves maybe not needed - it is form immune therapy maybe, so becaus of paracine signalling

Current Approaches to Heal Injured Vasculature

• Coronary Artery Bypass Grafting procedure invasive Vascular Access Graft
• Autologous vessel is choose for upper leg put in heart= internal throacic artery or saphenous vein, however are often not avaible in old people or made accessible for patients
• Synthetic in CABG is not available by nothing sticks, no trombosis, but also no cells + Patency: open of graft is very low.

What We Want in New Approach Healing Vasculature

• Small diameter targeted vasculature
• Patient specific diameter
• Immediate use and high primary patency
• Low thrombogenicity to be infection resistent which supports a long lifespan
• Long and difficult list and is needed for vascular tissue engineering
• Goals is to assemble functional construct that restore, maintain or imporve damaged tissues or whole organs

In Vitro Approach for Vascular Tissue Engineering

• Paradigm using bioreactor culturing and variation on maturation
• Harvest cells, cell expansion, scaffold creation and cell seeding onto the scaffold by the bioreactor cultivation, and if allogeneic then the decellularization will occour before the implantation.
• Company Example:
• Already in phase III trial. Humacyte uses donors to isolate smooth muscle cells and bioreactor for 8 weeks+ prolferating of cells and making of ECM to have white blood vessel structure: trasnplant
• AV graft is used and no immune rejection occurs,also for trauma and vascular excess for hemodialysis
• Pros+cons: immediate functioning and regulated/controlled through tria, however can be structually damaging:
• (Cell source needed: Progenitor cells? Where can you find them? How do you isolate them? Not off the shelfand Hands on- labor intensive steps)

In Vivo Approach for Vascular Tissue Engineering

• Is biodegradable for vascular grafts
• Mold is created fabricated the implanted of scaffold to grow the vessel. To used the body as bioreactor to exlant (take out) vessel to than deceullarize and implant which leads to remodeling.

= Scar like structure with lumen used as vessel due to autologous structure and No endothelial cells

• Implant under skin: luminized structure of collagen.
• No functional tests.

PROS-CONS

• Autologous possibilities with using bodies regenerative however,requires regenerative time, not every step can be controlled and is not off the shelve, and 2 surgeries needed for the mold

In Situ Approach for Vascular Tissue Engineering

• Implant of a scaffold slowly is slowly to induced angiogenesis
• Immune driven regeneration in wound healing: scaffold needs to adhere and allow bloodflow and withstand pressure and be removed

Scaffold is to have a aligned timed degradation or tissue formation, so timing is key for succes

• no cell cultivation, can be manipulated and 1 surgery with is off the shelve

• Biodegration and angiogenesis is needed and be on the bodys regenrative

General Factors That Can Influence Functional Tissue Outcome (Which VTE)

• Scaffold properties and patients unique inherit factors
• Inherited factors can also be depended for the race and anatomical location

General Questions When Designing Your Own Scaffold

• Applicaiton?
• Disease context?
• Apporach?
• Readout?
• Tissue to created with the body?

Paracrine Signaling

• Secreted molecules diffuse locally and trigger a response in neighboring cells(more integrines)

Stem cell goes damages tissue and can secrete all kind of factors and regenration is stimulated from anti-inflammatory cytokines and Stem cel is gone again

Extracellular Vescicles

• Exosomes: Cytoplasm from vesicles secreted from multivesicular body + escort proteins involved in cargo selection and budding with the smallest vesicles inside
• microvesciles come derviced from Cytoskeletion (disrupted with snare proteins and content lipids, protein, Rna ect)
• apoptotic bodies come when cells a dying,and oncosomes from cancer cells

Paracrine Signaling

• Paracrine signalling is a form of communication to trigger response and change cells
• Damaged tissue: Stem cell heals to land through factors -

Research: fractionated medium with molecules, and spectrometry + extra cellular vesicles

Making of Exosomes

• Budding of. Intraluminar vesicles with proteins environment

Making of Microvesicles

• Exocytoskeleten

Content of EVs

• Protein, miRNA, mRNA, metabolites, membrane proteins, lipids - membranes

Filtering Vesciles Out of Cells

-spin down cells - apoptotic and micro vesicles removal -then Isolate with 10K and Exosomes with then in Pellet

Functions of EVs

-Ligand-receptor interaction + Downstream signaling through Direct fusion + Accumulation of vesicles or transcytosis in intersines

Clinical Application of Exosomes

• No cells and cancerous used

MSC exosome: Organs and Tissue. Lots of pepers vs models -> kidney -> translator

Comparing MSCs vs EVs

EV have different procedures for cells. EV productions is limited Regulations and dificulities store Dosing

Setting Up Experiment With EVs

First look at quality EV + characteristics and apply for EV.EV distributions ( kidney liver) injury with Organ interactions 1.Quality stems 2.Check the character. 3.Combine orgs

Pancreas

• 99% digestive 1 % hormones ( alpha beta ( glucose)). 1 billion cells

Diabetes

Decrease in beta cells types Type 2 from age 2/3 Type 1 immune system and young

Treatment Type 1 Diabetes

Glucose devices and insuline

Pancreas Transplantation

High risks -> also affect kidneys Transplanation is functional 1/3 organ donor Not a choice

Islet Transplantation

• Risk, complications, etc, less cuering rate than a severe diabetes and HLA sensitizations

Main 2 Downsides of Organ Transplantation

• Limited supply and lifelong supresion

Use of Adult Stem Cells Diabetes

Neogenesis, Injection into kidney of mice

Use of Pluripotent Cells Diabetes

Show c -petide by v12, is maturation which can Mimic -disease modelling,optimization VX needs mature longer

Overcome Need for Immune Supression When Using Stem Cells

• The solution seems to be the use of pluripotent stem cells Device -> implant

EUSTM

• time and cost
• TM Bench Benchside Timewlse

Translational Medicine

• Combined -> Bench bedside - close model costs money. 1000 will ->

Translational Cardiac RM

• Preclinical research and cardiac

How Improve Quality of Pre-Clinical Research

Models need to improve

Optimizing Large Animal Models

• Meta analzye to show and know

Retention of BM-MSC in Chronic MI (Myocardial Infarction) in Pigs

• No significant difference in retention for different delivery routes. Less invasive route therefore advisable.

Growth Factor + Hydrogel: Pig Study Cardiac

• Injected via cathethers to target specific area. Gel solidifies after injection and provides controlled prolonged release and availablility of growth factors. Prevent off-target effects and limit wash out.

Tissue Engineering Pig Study Cardiac

Injection of shape-memory engineerd

Gene Therapy Pig Study Cardiac

• PLN -> models and deliver

autotransformation -> Moon Shot ( take heart of patient and back) Healthy and do bio

Pig Studies Examples (4) (Onderzoeksrichtingen)

BM-MSC and 4) Tissue

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Large Models Advantages and Disadvantages

• Advantages and disadvatges of pysholgy and disease Pig = promising + translate
• Costs

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Myocardial Infarction

Cell do not -> fribotics Theapy will heal

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Cardiac Regenerative Medicinal Products Generations

regenration effectes

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Use of Cardiac Progenitor Cells

Lab -> animal = pump

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Use of Microtissues for Cardiac Regeneration

Higher signal in microsheres and VASC

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Use of 3D Printing for Biocomplexes (Cardiac Regenration)

High survival

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Use of iPSC Cells Un Cardiac Regeneration

Washout but safety issue Direct inject +add

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Cardiac Patch Made of a Hydrogel (Collagen) and iPSC-CMs

Challneges:Tissue and Myo

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Cardiac Tissue Engineered Patches 3D Printing

Inprove for.

Add. Laser vascular

Role Fibroblasts Iin Combination with iPSC-CMs

iSP -> denser

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Hypoimmunogenic iPSCs

• Hypoimmucene ->