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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!

Goals of Thesis

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

  • ~500 cells in a gravity well
  • excellent for toxicity assay and discovering dose dependency (i.e. 100 nM vs 100 µM)

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

  • Steve Kattman (CDI),(Gordon Keller Lab) discovered in 2006 and 2011 publications
  • Temporal expression of Activin/Nodal pathway and BMP control cardiac progenitor 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+

  • bioreactor needed
  • 10 million to 125,000

The battle we lost

Survivors are left with a broken heart

  • Myocardial Infarct weakens the wall of the heart, cardiomyocyte death
  • Intrachamber pressure increases due to weakened ejection fraction
  • Heart Rupture - patient death

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

  • Its a complex experiment but ultimately well documented and thoroughly published

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

  • Death Valley National Park
  • Sequoia National Park
  • Pacific Crest and the Continental Divide Trails (work on the big three)

60 beats

Professionally:

  • Stem cell culturing, mentoring and teaching basics
  • Synthetic Blastema for limbs (S. Gilbert)
  • Cell therapies for regenerative medicine
  • Expand my differentiation skills to new cell types.
  • PhD in the next five years

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 am a weekend warrior
  • Worked alone extensively
  • All the orders, solutions/dilutions, lab maintenance, and experiments fell on me, at one time or another
  • I have had my hands very dirty and very sterile in the name of research
  • Taught in five graduate courses
  • Worked in 7 labs in three countries

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

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