Conversion of Adipogenic Mesenchymal Stem Cells into Mature Cardiac Myocytes (Cardiac Myocytes) - 09.27.17

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Conversion of Adipogenic Mesenchymal Stem Cells into Mature Cardiac Myocytes (Cardiac Myocytes) uses the microgravity environment of space to examine how stem cells differentiate into specialized heart cells (cardiac myocytes). Previous studies using microgravity chambers on Earth have found that low gravity environments help specially programmed stem cells move towards becoming new heart muscle cells. The Cardiac Myocytes experiment delivers frozen stem cells in an experimental setup to the International Space Station where the cells are thawed, cultured under specific conditions, tagged and then returned to Earth for analysis and comparison with control batches.
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The following content was provided by Robert J. Schwartz, Ph.D., and is maintained in a database by the ISS Program Science Office.
Experiment Details

OpNom: Cardiac Myocytes

Principal Investigator(s)
Robert J. Schwartz, Ph.D., University of Houston, Houston, TX, United States

Co-Investigator(s)/Collaborator(s)
Bradley McConnell, Ph.D., University of Houston, Houston, TX, United States
Ravi Birla, Ph.D., Texas Heart Institute, Houston, TX, United States
Dinakar Iyer, Ph.D., University of Houston, Houston, TX, United States
Clifford C. Dacso, M.D., M.P.H., Baylor College of Medicine, Houston, TX, United States

Developer(s)
Techshot, Inc, Greenville, IN, United States

Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)

Sponsoring Organization
National Laboratory (NL)

Research Benefits
Scientific Discovery, Earth Benefits

ISS Expedition Duration
April 2017 - February 2018

Expeditions Assigned
51/52,53/54

Previous Missions
Information Pending

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Experiment Description

Research Overview

  • Ground studies for Conversion of Adipogenic Mesenchymal Stem Cells into Mature Cardiac Myocytes (Cardiac Myocytes) are complete and found that a pair of human transcription factors, ETS2 and MESP1, converted human fat stem cell into cardiac myocytes that were immature.
  • These factors up regulated cardiac mesoderm cell surface markers and a cadre of cardiac regulatory factors, but without the appearance of many calcium handling proteins needed for electrical conductivity.
  • The role of three-dimensional (3D) cardiospheroids was also explored and found to form in a rotating Synthecon bioreactor that mimics zero gravity on earth. Without the addition of any other reagent or drug the robust induction of adult myosin heavy chains, contractile sarcomeres, ion channels, calcium handling channels and pumps in 3D cardiospheres was observed.
  • The gene expression network of ion channels, SR and t-tubules genes was decoded using cutting edge genetic informatics on the ground experiment and found the appearance of many genes involved with calcium handling and myocyte maturation.
  • The experimental paradigm contributes to a novel regenerative strategy that enhanced myocyte maturation occurred from converted human fat stem cells. This first step demonstrates the efficacy of the microgravity environment for preparation of robust platforms for organ repair.
  • Cardiac Myocytes hypothesizes that the bioreactor may mimic the microgravity experience but certainly does not fully duplicate the complete loss of gravity, as on the International Space Station (ISS).
  • Cardiac Myocytes flies the cardiac progenitor cells (CPCs) in a Techshot Bioreactor on the ISS and evaluates myocyte maturation under microgravity conditions.
  • The metabolics and calcium handling properties of the reprogramed myocyte flown in space are then tested for their improved potential for repairing a damaged mouse heart model on earth.
  • Thus, research sponsored by the center for the Advancement of Science in Space (CASIS) supports the first conversion of human adipose-derived mesenchymal stem cells (ADMSCs) into cardiac myocytes. Here, a novel maturation paradigm is created by culturing 3D cardiospheroids that enhances electrical maturation of the cardiac myocytes. Finally, the reparative capacity of CPC’s differentiated from ADMSC’s is critical for clinical applications on earth.

Description

The human adult adipogenic (fat) mesenchymal stem cells (hADMSCs) are reprogrammed to form cardiac progenitors (CPCs) in bulk culture and are infected in the Schwartz laboratory to carry a myosin light chain 2V-Green fluorescent reporter and a luciferase constitutive vector to produce light emissions, important for following reprogrammed cells injected into infarcted hearts. Also, the alpha myosin heavy chain (αMHC)‐Cherry lentiviral vector reporter is infected into pre-reprogrammed cells to follow their terminal differentiation of reprogrammed cells following three-dimensional (3D) cardiosphere formation. On the ground, hADMSCs is induced to transform into cardiomyoctyes within the Techshot CellCult 50 mL rotating wall bioreactor. Within this microgravity mimic bioreactor, these cells aggregate and continue to grow forming 3D cardiospheres. At three time points following rotation in the bioreactors early (3 days), middle (5 days) and late stage (7 days) cardiospheres are harvested and frozen for earth bound studies. A portion of these frozen cardiospheres are assayed for changes and evaluated for their ability to mature and activate the fluorescent adult MHC6 mCherry reporter. These mature myocytes are used in rescuing the damaged heart. The murine cardiac infarct model is an excellent test system to evaluate the maturation, differentiation, integration and survival of the reprogrammed human myocytes generated on the International Space Station (ISS). Since, ETS-MESP1 myocytes is influenced by forming 3D cardiospheres will maturation of Reprogrammed human myocytes under zero gravity support cardiac repair?
 
Two identical Techshot CellCult units are prepared with culture medium and placed in cold stowage for flight to the ISS. Once on orbit, the CellCult cassettes are simultaneously placed within the Techshot Advanced Space Experiment Processor (ADSEP) facility and allowed to come to temperature. Once at operating parameters, the frozen cardiospheres are injected directly into each CellCult cassette’s rotating bioreactor and incubated. Samples are allowed to grow at 370C while taking multiple time point samples (15 mL each) 3three times during flight. Reprogrammed myoctes (approximately 2.5 X 106/50 mL) are cultured in media, aMEM, 5% Horse Serum (and or 10% fetal bovine serum) 1% antibiotics, 100 uM Norepinephrine stored in ascorbic acid) and sampled three3 times starting from day 2 through day 7. Fresh medium already resident within the bags in each CellCult cassette replenishes volumes taken during sampling, with the ability to change an additional 100% of the volume automatically at fixed intervals during the duration of the experiment (single bolus or multiple increments). These time point samples are placed in a 10 mL capped sample tube and concentrated within the high speed centrifuge. Every time point sample is resuspended in cryopreservant (5% DMSO, 20% Fetal Bovine Serum, 75% aMEM) and then transferred to MELFI (-80°C) until return to the Schwartz lab for further analysis on Earth. The cryopreserved cells are assayed for calcium handling properties and reparative activity in a SCID mouse cardiac infarct model.
 
In comparison, myocytes are grown in a Synthecon bioreactor and Techshot CellCult units which contains 50 mL of media (aMEM, 5% Horse Serum, 1% antibiotics, 100 uM Norepinephrine/stored in ascorbic acid)) which is slowly rotated (10 cycles/min.); thus, resembling the Synthecon bioreactor of the earth bound experiments. Sampling is done every other day and microscopic imaging are likely to show live myocyte 3D spheroids, that fluoresce the myosin heavy chain reporter mCherry. Frozen samples are retrieved, thawed and injected in to the bioreactor containing media. Samples are allowed to grow at ambient temp while taking a small volume of time point samples three3 times. These time point samples are placed in a sample tube containing cryopreservant, spun down then placed in MELFI (-95°C).
 
On Earth, the researchers Fluorescence-activated Cell Sorting (FACS) -sort out reprogrammed cells and analyze them with a Kinetic Image Cytometry (KIC) instrument which records images at a frequency of 33 Hz using CyteSeer software. Cells are plated at 15,000 cells/well onto gelatin coated 96-well plates. Each plate has at least 8 experimental wells that contain repeats of a test ETS factor-Mesp1 doxy-induced cardiac progenitors (mCherry positive) and 8 negative control wells for plate Z’ determinations to record each plate’s dynamic range and ensure acceptable drift of dynamic range (<20% signal/background) across all plates. Control wells are also used for inter-plate normalization. The software-assisted image segmentation into individual cells monitors many individual spontaneously contracting cells for fluorescence signals from Fluo-4 as an intracellular calcium (Ca2+) indicator, major histocompatibility complex (αMHC-Cherry) reporter as an indicator of cardiac development and DAPI for nuclei.
 
The genetic readout of individual ion channels, SR and t-tubules are assayed from RNA samples taken from drug treated cells and assayed by quantitative PCR assays. A heat map of gene expression is generated from cardiac myocytes generated from murine ES cells and measured the gene activity of at least 32 relevant ion channels, SR and t-tubule genes. Comparisons of gene activity is made against Ets2/Mesp1 induced cardiomyocytes formed in 3D cardiospheres by profiling their expression of ion channel/pump genes. The genetic readout of individual ion channels, SR and t-tubules are assayed from RNA samples taken from 3D cardiospheres by quantitative PCR assays or by high.
 
The data is shared when the GeneLab project is operational. The RNA expression heat maps are compared to look for regulatory coordination between the formation of 3D cardiospheres and gene readout. Comparisons of gene activity is made against Ets2/Mesp1 induced cardiomyocytes in 3D cardiospheres, by profiling their expression of ion channel/pump genes. Isolated RNA is used to generate small fragments of cDNA suitable for sequencing using the Illumina NextSeq 500. Raw sequencing reads are mapped to the transcriptome using BLAT (BLAST-like alignment tool) and then tag counts are summarized and normalized using ERANGE and DESeq software. Since, ADMSCs do not appear to express cardiac restricted genes, it is highly likely that the temporal appearance of primary ion channels, SR, and t-tubules transcripts are dependent upon transcriptional activation. The early appearance of epigenetic histone marks, H3K4Me3, H3K27Ac and H3K36Me3 that denote the activation of downstream gene targets are also examined. Up-regulated ion channels, SR, and T-tubules target genes display stronger H3K4me3 enrichment at their promoters and increased chromatin remodeling by H3K27Ac throughout the transcribed gene and enhancers and transcriptional elongation by H3K36Me3. The alignment profiles of ChIP-Seq data from at least 2 different anti H3K4Me3 are required to show a Pearson correlation coefficient of 0.9 or better. A peak height of ≥5 is expected; and a FDR = 0.015 when compared to multiple control ADMSC cells samples, a stringent FDR in ChIP-Seq studies. The widely used MACS2 (18) is applied to the data with a stringent FDR cutoff of 0.01. It is expected that control ADMSCs do not show H3K4Me3 or H3K27Ac enrichment for many of the ion channels, SR, and T-tubules target genes.
 
The results of the reprogrammed ETS-MESP1 CPCs are compared to those matured under zero gravity conditions on the ISS and in the bioreactors on earth. An ultimate test of the reprogrammed human myocytes is their ability in rescuing the damaged heart. The murine cardiac infarct model is an excellent test system to evaluate the maturation, differentiation, integration and survival of our reprogrammed human myocytes generated on the ISS. Since , ETS-MESP1 myocytes is are influenced by forming 3D cardiospheres does maturation of rReprogrammed human myocytes under zero gravity support cardiac repair? The hypothesis is reprogrammed human myocytes restore cardiac function in a cardiac infarct model because of cardiac maturation of calcium handling proteins.
 
To positively identify the injected cells in the heart, ETS-MESP1 myocytes are permanently labeled while in culture using a lentiviral luciferase reporter pCMV-Luc infected just prior to cardiac injection. The bioluminescent luciferase marker allows for imaging localization and tracking in vivo, using the Kodak Molecular Imaging System for small live animals located at the University of Houston. In addition, the highly fluorescent viral MHC6-mCherry reporter gene, which was used in the Ets2/Mesp1 reprograming studies, is infected into NHDFs prior to reprogramming. These markers are also detected during histological analysis by microscopy, following myocardial functional studies.
 
A mouse model of ischemic MI in male C.B-17 SCID mice is induced by chronic ligation of the left anterior descending (LAD) artery standardly done by Dr. Bradley McConnell at the University of Houston. Ligation is verified by immediate myocardial blanching and anterior wall dysfunction. All animals with anterior wall motion dysfunction consistent with the infarct are randomized into the various groups. ETS-MESP1 myocytes are injected, using five5 2 uL injections (total volume = 10 uL) each with 20,000 YFP cells (total number of cells = 0.2 x106 cells) into the infarct and border zone areas, immediately following LAD ligation. Controls receive PBS only and/or untreated human adipogenic stem cells. and experimental groups. Following cellular injections, the chest is closed and the animals monitored closely. Cell tracking of the luciferase marker in mouse hearts is imaged live using bioluminescence at 24-hr post cell injection.
 
To determine and monitor cell engraftment and survival, hearts are imaged live again once a week during the course of the 2-week and 4-week assessment plans. The number of injected cell number versus bioluminescence signal is evaluated for estimation of cell survival and cell proliferation. To determine and monitor cell engraftment and survival, hearts are imaged in vivo once a week during the course of the 2-week and 4-week assessment plans. The number of injected cells versus bioluminescence signal is evaluated for estimation of cell survival and cell proliferation. To identify transplanted human ETS-MESP1 myocytes in the mouse heart, a monoclonal fluorescence antibody that reacts with the human major histocompatibility complex (αMHC) class I, HLA-A, B, C that are expressed by all human nucleated cells is used. The monoclonal antibody (clone W6/32) has been successfully used in previous studies to identify human cells in mouse heart. To examine if the injected cells develop into cardiomyocytes, heart sections are double stained with both fluorescent anti-HLA-ABC antibody and anti-cardiac troponin T conjugated with a fluorophore of different emission wave length. Endothelial cells derived from the reprogrammed cells are recognized by staining the tissue section with anti-HLA-ABC and anti-VE cadherin. A fluorophore-conjugated anti-smooth muscle alpha-actin is paired with anti-HLA-ABC to examine smooth muscle cells. To determine if the transplanted cells develop into cardiomyocytes that are engrafted in the heart and form gap junctions with the host cardiomyocytes, an antibody reacts with connexin 43, an important adhesion molecule that is thought to have a crucial role in the synchronized contraction of the heart is used together with anti-cardiac troponin and anti-HLA-ABC to stain the heart sections. The heart sections are then examined under an epi-fluorescence microscope.
 
Myocardial functional assessment of the CPC-treated mice is done by echocardiography, electrocardiogram and hemodynamics. (1) Transthoracic echocardiography is performed at BCM Phenotyping Core Facility using a Vevo 770 high-resolution micro-imaging system under anesthesia to evaluate cardiac function and dimensions at 24-hr post-MI and at 1, 2 and 4-week intervals post-MI. 2-D guided M-mode images of the left ventricle (LV) is taken using the parasternal-axis view at the level of the papillary muscles. Left ventricular fractional shortening (LVFS), ejection fraction (EF) and LV mass is calculated from wall and chamber dimensions. (2) Electrocardiograms (ECGs) are performed 1, 2 and 4-week post-MI on conscious animals without restraints from all four extremities. (3) Hemodynamics are evaluated with the MPVS Ultra (Millar Instruments, Inc.) to measure real-time LV P-V relationships as LV systolic and diastolic properties are measured in Dr. McConnell’s laboratory with a 4-electrode P-V 1.0-French catheter within the mice LV. P-V loop measurements are done using closed- and open-chest techniques to decrease preload before and after isoproterenol or dobutamine infusion.
 
The expected LVFS without induction of heart failure is 60% (SD=6.3%) (22,23). Assuming the injection of stem cells, animals have a LVFS of 70 ± 6.3%, in the absence of heart failure induction, the sample size is 9 mice/group with 90% power (SD=6.3%) with one factor ANOVA using 2-tailed alternatives and a type-1 error level of 0.05. Additional 5 mice/group are added for the expected surgical mortality rate and an additional 2 mice/group are added for unexpected events. Statistical methods include chi-square, logistic regression and ANOVA methods for cardiac and biodistribution studies with Tukey’s post-hoc for p-values ≤ 0.05.
 
It is anticipated that the hearts injected with the ETS-MESP1 myocytes grown as 3D cardiospheres immediately following a myocardial infarction (MI) improves cardiac function by reduced arrythmias. Hearts similarly injected with the mature myocytes with improved calcium handling likely improve cardiac function by enhancing CM survival and improved cardiac remodeling and cardiac contractility.

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Applications

Space Applications
This project demonstrates how space can be used to achieve medical breakthroughs. Cardiac Myocytes capitalizes on the specialized environment of space to bridge a critical gap in understanding of stem cell development. The experiment also demonstrates ways for using space as a production facility for vital medical therapies. In addition to these contributions, Cardiac Myocytes advances understanding of regenerative therapies that can be used to counteract muscle and bone loss suffered by astronauts during long term space travel.

Earth Applications
Heart failure is a major health risk and medical expense on Earth. Cardiac Myocytes tests cellular pathways for therapies that can help repair hearts after heart attacks. Using the specialized conditions of space in guiding stem cells to become healthy heart cells can help reduce the risk of heart failure and other diseases.

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Operations

Operational Requirements and Protocols

Once at operating parameters, the frozen cardiospheres are injected directly into each CellCult cassette’s rotating bioreactor and incubated. Samples are allowed to grow at 37°C while taking multiple time point samples (15 mL each) three times during flight. Reprogrammed myoctes (approximately 2.5 X 106/50 mL) are cultured in media, aMEM, 5% Horse Serum (and or 10% fetal bovine serum) 1% antibiotics, 100 uM Norepinephrine stored in ascorbic acid) and sampled three times starting from day 2 through day 7. Fresh medium already resident within the bags in each CellCult cassette replenishes volumes taken during sampling, with the ability to change an additional 100% of the volume automatically at fixed intervals during the duration of the experiment (single bolus or multiple increments). These time point samples are placed in a 10 mL capped sample tube and concentrated within the high speed centrifuge. Every time point sample is resuspended in cryopreservant (5% DMSO, 20% Fetal Bovine Serum, 75% aMEM) and then transferred to MELFI (-80°C) until return to the Schwartz lab for further analysis on Earth. The cryopreserved cells are assayed for calcium handling properties and reparative activity in a SCID mouse cardiac infarct model.

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Decadal Survey Recommendations

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Results/More Information

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Imagery