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In Vitro Methods to build the Cardiomyocyte Microenvironment
3 Models to generate Cardiomyocyte Drug Discovery Platform
Methods Time
Problem: How do we test cardiogenic drugs in a risk free environment
Three things to repair a heart
1. cardiogenic drugs (transcription mimics)
2. endogenous transcription factors
3. mechanotransduction and assimilation into SAN pace!
Drug Discovery
In simplest terms my goal:
1. Design a pipeline which increase in specificity of cell type and population towards native heart
2. Design a pipeline which becomes more and more like the native heart microenvironment
3. 6 months to complete research
Design a pipeline for screening molecules for the 3i Regeneration project;
Three models:
1. Hanging Drop
2. Directed Differentiation
3. MACS Mesoderm specific
Explore cardiomyocyte generation and production through the lens of: regenerative medicine, drug discovery and developmental biology
Embryoid Body formation via Hanging Drop
Its an honor to be here
Alexander R. March
Tervetuloa!
1. M.Sc. Molecular Biotechnology
Minor: Genetics
University of Helsinki
Welcome!
2. B.Sc. Microbiology
Minor: Biochemistry and Molecular
Biology
Certificate in Biotechnology
University of Massachusetts Amherst
Directed Differentiation
(Figure courtesy of 3D Biomatrix)
Methods in Functional
Genetics and Development:
First did this 3 years ago, taught the course the following year
a younger man
Cardiomyocyte purity from an EB molecule assay. A selection of molecules from flow cytometry results from a Spontaneous Differentiation Molecule Assay with molecules present from Day 2-4 and Day 6-10. Flow cytometry on Day 10. Flow Cytometry on the BD Accuri C6 sampler with 10,000 cells counted as the minimum threshold. N=3, error bars (SEM)
spontaneous differentiation
Why build a heart in the lab?
-Magnetically Activated Cell Sorting (Miltenyl Biotech)
-Sort directed differentiation for FLK1 and PDGFRa
-Yields a Multipotent CPC population (FLK1+) and (PDGFRa+)
-Cell Surface Markers CD309 receptor VEGFRa (FLK1) & CD144a (PDGFRa)
Outline:
I. Introduction to Myocardial Infarction
II. Regeneration Vs Repair in the left ventricle
III. Masters thesis research in cardiogenic Drug Discovery
Title: Molecular Mechanisms of Multipotent Cardiac Progenitor Cell Differentiation, Drug Discovery and Signaling Pathways
32.4 million myocardial infractions every day on Earth (WHO)
MI is a result of blockage in the Coronary artery (CHD)
Oxygen starvation in the peripheral tissue, i.e. left ventricle
Mass die off of cardiomyocytes directly proportional to time
GFP-Myl2
ventricular cardiomyocytes
Myosin regulatory light chain 2
Too few double+
Survivors are left with a broken heart
Regeneration vs. Repair
Do endogenous CPC exist in the adult heart?
Cardiac Fibroblasts save the day?
Adult heart cannot regenerate from trauma (so far)
Beating Cardiomyocytes can be made
Scar tissue and static remodeling
1% per annum turnover
Like having a marble in your heart
My contribution
How do we turn this on?
Within the developing heart, embryonic cells traverse a carefully choreographed Waddington's landscape where the spatial and temporal expression of cardiac gene cassettes drives cells to their specialized fates
"If we can get even 5% regeneration! A patient could walk to the bathroom again without losing their breathe" -Bogac Kaynak (University of Helsinki)
Directed Differentiation
attempts to mimic this development
We don't necessarily need a total win!
The Life of the Heart
Tested over 100 novel compounds
Hoping for publication next year in Pharmaceutical Chemistry
Patent of compound C1
Research Funded by: Finnish Heart Foundation, Tekes, 3i Regeneration, Faculty of Pharmacology and Pharmacotherapy
heart en route to patient
The heart pumps all the oxygenated blood, plasma, and nutrients for all the organ systems of the adult vertebrate
And it all starts on Day 22, as a linear tube
Beating uninterrupted from E20-E22 until the end of life
My Goals for the lab
To join the USC Keck Community
UCLA Medical School
Human heart lasts 6 hours on ice
Many never make it to patients
Personally:
Hike every mountain
60 beats
Professionally:
resting Heart 58 Bpm
26 years old - 792,604,800 beats so far
Questions, Comments
&
Anything in between
Key References & Highlight Articles
Kattman SJ, Huber TL, Keller GM. Multipotent FLK-1+ Cardiovascular Progenitor Cells Give Rise to the Cardiomyocyte, Endothelial, and Vascular Smooth Muscle Lineages. Developmental Cell; Elsevier. 2006 Nov. vol. 11. pg. 723-732.
Kattman SJ, Witty AD, Gagliardi M, Dubois NC, Niapour M, Hotta A, Ellis J, Keller G. Stage-Specific Optimization of Activin/Nodal and BMP Signaling Promotes Cardiac. Cell Stem Cell. 2011 Feb., vol. 8, pg. 228-240.
Martin-Puig S, Wang Z, Chien KR. Lives of a Heart Cell: Tracing the Origins of Cardiac Progenitors. Cell Stem Cell; Review. 2008 April. vol. 2. pg. 320-331.
Kurokawa YK, George SC. Tissue engineering the cardiac microenvironment: Multicellular microphysiological system for drug screening. Advanced Drug Delivery Reviews; Elsevier. 2015. ADR-12819
Später D, Hansson EM, Zangi L, Chien KR. How to make a cardiomyocyte. The Company of Biologists Ltd; Development. 2014. vol. 141. pg. 4418-4431.
WHO. Global Health Observatory data repository; Causes of Death by Numbers of Deaths. World Health Organization. 2008.
<http://apps.who.int/gho/data/node.main.CODWORLD?lang=en>
stay active!
How I fit?
All data presented is property of Alexander R. March, unauthorized reproduction is not allowed
Background Basics Cardiomyocytes In the Heart:
No lab is run or operated alone. Having a competent team means working together and moving forward every day of the week!
I. Cardiomyocytes are individual cells that comprise the heart myocardium. (The cells that beat)
1. From E20-22 till death do us part
2. Cardiomyocyte make up ~90% of total cell mass of the heart (Zhang 2015)
3. Binucleated muscle cells, terminally differentiated, rod shaped, sacromere rich, cTnT positive
Temporal Expression is the key, Directed Differentiation utilizes 40 - 43 hour induction
CPC:
FLK1+
Islet1+
Sca+
Bry+
PDGFRa+
Mesp1+
Cardiomyocyte:
cTnT+
Kattman Found FLK1+ CPC marker (E4.25) by accident in a hunt for Hematopoietic progenitor (E3.25)
Martin-Puig 2008
Can we ID a true CPC and lock its
fate control