Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

Podsumowanie

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

Streszczenie

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs).

We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.

Wprowadzenie

The heart is the first functional organ to develop in the embryo^1,2. In conjunction with the circulatory system, it supplies oxygen, nutrients, and a waste disposal mechanism during development. Three weeks after fertilization, the human heart beats for the first time and its proper regulation is maintained by cardiomyocytes (CMs). The irreversible loss of these specialized cells is therefore the fundamental issue underlying progressive heart failure. While some organisms such as the zebrafish and Xenopus have the potential for cardiac regeneration, the adult mammalian heart is more limited^3,5,6. Thus, given the critical function of the heart, it is not astonishing that heart disease is the leading cause of death in the world, accounting for 600,000 deaths in the United States alone⁷. Therefore, cell-based therapies to efficiently repair or replace the injured myocardium are of great clinical interest.

The seminal study of Yamanaka and colleagues⁸ showed that forced expression of four transcription factors is sufficient to convert fully differentiated fibroblast cells to pluripotent stem cells. However, the tumorigenic capacity of all pluripotent stem cell strategies has been a critical concern in their use for therapeutic purposes. This motivated the scientific field to search for alternative methods to transdifferentiate cells while avoiding a pluripotent stage. Recently, several groups have shown the feasibility of this strategy by displaying direct conversion of mouse fibroblasts to induced cardiomyocyte-like cells (iCLMs) with the ectopic expression of the transcription factors Gata4, Mef2c, Tbx5, and later on, Hand2 (GMT and GHMT, respectively)^9,10. Furthermore, the same strategy can be performed in vivo and in human-derived tissues^9,11,12. Recent studies have highlighted additional factors or signaling pathways that can be modulated to further improve cardiac reprogramming efficiency^13,14,15. Taken together, these studies demonstrate the potential of directed transdifferentiation for regenerative therapies. However, the low efficiency of CM reprogramming, the unknown molecular mechanisms, inconsistent reproducibility due to methodological differences¹⁶, and the heterogeneous nature of iCLMs remain unaddressed.

In order to directly evaluate iCLM heterogeneity, we designed a discrete and robust single-cell assay for the identification of sarcomere development and cardiac lineage specification-two necessary characteristics of functional cardiomyocytes. There are at least three major types of CM in the heart as defined by their location and unique electrical properties: atrial (AM), ventricular (VM) and pacemaker (PM)^17,18,19,20. In an orchestrated combination, they allow the proper pumping of blood. During heart injury, one or all subtypes might be affected, and the type of cell therapy would need to be addressed on a case-by-case basis. Currently, most strategies focus on the overall generation of cardiomyocytes, while little work is being done to study the molecular mechanisms that regulates subtype specification.

The following study details how to properly quantify well-organized sarcomeres and identify a diverse set of cardiomyocyte subtypes. Using a pacemaker (PM)-specific reporter mouse, we are able to apply an immunocytochemical approach to distinguish induced atrial-like myocytes (iAM), induced ventricular-like myocytes (iVM), and induced PM-like myocytes (iPMs)²¹. Based on our observations, only cells that exhibit sarcomere organization are capable of spontaneous beating. This unique reprogramming platform allows for assessing the role of certain parameters in sarcomere organization, subtype specification, and efficiency of CM reprogramming at single-cell resolution.

Protokół

All experimental procedures involving animal practices were approved by the Institutional Animal Care and Use Committee at UT Southwestern Medical Center.

1. Isolation of Hcn4-GFP E12.5 Mouse Embryonic Fibroblast (MEFs)

1. Set up timed matings between homozygous Hcn4-GFP males and CD-1 females.
2. Sacrifice pregnant female at E12.5 by carbon dioxide euthanasia and subsequent cervical dislocation.
   1. Remove uterine horns with dissecting forceps as described previously^22,23, and place them into a Petri dish on ice with 1x phosphate-buffered saline (PBS) without Ca²⁺ or Mg²⁺.
3. Perform all subsequent steps in the tissue culture hood using sterile technique.
   1. Remove the embryos from the uterus and amniotic sac using scissors and dissecting forceps. Keep the placenta attached for better handling. Pregnant CD-1 females typically give birth to between 10 and 14 pups.
   2. Using dissecting forceps, take the isolated embryos and quickly rinse them twice in 70% (v/v) EtOH.
      NOTE: The washes should be fast to minimize cell death.
   3. Remove the head, limbs, tail, and internal organs, including the heart from the isolated embryos.
   4. Place the remaining tissue in a 10-cm dish with 1 mL of 1x PBS and finely mince using a sterile razor blade to approximately 1 mm³ in size.
   5. Transfer minced tissue into a 50 mL conical tube with PBS.
   6. Spin at 300 x g for 3 min. Carefully aspirate excess PBS.
   7. Add 1 mL of sterile 0.25% trypsin-EDTA per embryo. Incubate cells in a 37 °C water bath for 15 min. Gently mix the tube every 4 min. Over-digestion of the tissue will significantly lower the yield.
   8. Vortex cell mixture at maximum speed (3,200 rpm) for 4 s.
   9. Add 2 mL of fibroblast media per embryo and mix. Filter through a 100 µm cell strainer using a pipette to aid the cells through the strainer. Refer to Table 1 for the formulation of all subsequent mediums.
   10. Spin at 300 x g for 4 min. Carefully aspirate supernatant.
   11. Add 10 mL of fresh fibroblast media per 3 embryos and triturate 6-10 times.
   12. Plate the cells in 1 15-cm tissue culture dish for every 3 embryos prepared. Culture overnight in a 37 °C, 5% CO₂ incubator.
   13. After the overnight incubation, replace the media with fresh 30 mL of media per plate. Place cells back into the incubator overnight.
       NOTE: Check for Hcn4-GFP⁺ cell contamination under a fluorescent microscope. The culture should be GFP^- and only become GFP⁺ upon reprogramming.
   14. The next day, harvest cells with 3 mL of fresh pre-warmed 0.25% trypsin-EDTA. Count and freeze cells. Typically, freeze cells at 3 x 10⁶ cells per mL. The expected yield should be 3 x 10⁶ cells per embryo.

2. Retrovirus Production and Reprogramming

Caution: The following protocol requires production and handling of infectious retroviruses. Perform the following steps in a Biosafety Level 2 cabinet under BSL-2 guidelines and sterile technique. Use 10% bleach to dispose of all materials exposed to retroviruses.

1. Retrovirus production and MEFs preparation
   NOTE: The following protocol is for retroviral production in 6-well plates and MEF infection in 24-well plates. For other formats, refer to Table 2. MEFs are plated at Day -1, so the timing will need to be coordinated appropriately for each experiment (Refer to section 2.3 and Figure 2).
   1. Maintain Plat-E (PE) cells as per manufacturer's recommendations. Briefly, culture PE cells in DMEM supplemented with 10% FBS, 1 µg/mL puromycin, 10 µg/mL blasticidin, penicillin, and streptomycin. Passage cells 1:4 every two days when the culture reaches 70-90% confluency.
   2. Day -2: The day before transfection, seed Plat-E cells at 1 x 10⁶ cells/well on a 6-well plate in transfection media. Cells should be 70-80% confluent at the time of transfection.
2. Transfection using a commercial transfection agent.
   NOTE: The commercial reagents should be at room temperature (RT) before transfection. For the DNA transfection, add each retroviral plasmid DNA individually (G, H, M, and T) to form a GHMT cocktail⁹.
   1. Day -1: In a 15 mL conical polystyrene tube, mix 60 µL of reduced serum media with 6 µL of transfection reagent per reaction for a 6-well plate format. Incubate the mixture for 5 min at room temperature.
      NOTE: Since the transfection reagent used here binds to plastics, add directly to the reduced serum media to avoid any decrease in transfection efficiency.
   2. Add a total of 2 µg of GHMT cocktail per reaction and gently tap to mix it. Do not vortex. Incubate the reaction for 15 min at RT.
   3. Add the mixture from step 2.2.3 to the PE cells in a drop wise manner.
   4. Incubate the transfected Plat-E cells overnight in a 37 °C, 5% CO₂ incubator. Record the time of transfection.
3. Seeding of Hcn4-GFP mouse embryonic fibroblasts.
   1. 1 h before plating MEFs, prepare a 24-well plate for immunocytochemistry.
      1. Add a 12 mm fibronectin coverslip per well.
      2. Coat wells with 300 µL of bovine collagen solution (e.g., SureCoat) and incubate in a 37 °C incubator for 1 h.
      3. Aspirate coating solution immediately before MEF plating.
   2. Thaw a frozen vial of Hcn4-GFP MEFs and wash x1 with pre-warmed fibroblast media at 500 x g for 5 min.
   3. Determine cell viability using trypan blue exclusion or similar dyes. Calculate the number of viable cells per mL of culture using the following formula:
      % viable cells = [1.00 - (Number of blue cells/ Number of total cells)] x 100
      1. Calculate total number of viable cells using the following formula:
         Viable cells = % viable cells x dilution factor x 10,000 x total volume of cell suspension
   4. Seed 3 x 10⁴ cells per well onto a 24-well plate with previously prepared bovine collagen solution-fibronectin coverslip.
4. Transduction and reprogramming of MEFs
   NOTE: According to the manufacturer's notes, properly maintained Plat-E cells produce an average titer of 1 x 10⁷ infection units/mL. Although the titer is not directly measured for each experiment, a GFP control is always included as a surrogate for infection efficiency. High GFP expression and intensity (GFP⁺ > 95%) typically correlates with successful GHMT-mediated generation of iCLMs.
   1. Day 0: 24 h post-transfection, filter the PE retroviral medium through a 0.45 µm-pore size surfactant-free cellulose acetate filter and transfer to a 15 mL conical tube. Add polybrene to a final concentration of 8 µg/mL. Carefully replenish cells with 2 mL of fresh transfection medium.
      NOTE: Cells easily detach from the plate if media is changed too rapidly.
   2. Aspirate the medium of the cultured MEFs and add the freshly collected retroviral medium; it should yield 1.7 mL of media per well of a 6-well plate. Add ~800 µL per well of a 24-well plate. Return MEF plate to the incubator and incubate overnight.
   3. Day 1: Repeat steps 2.4.1 and 2.4.2. Discard cells after the 2^nd virus collection. Return infected MEFs to the incubator and let them rest overnight.
   4. Day 2: 48 h post-induction, aspirate the cell conditioned media and wash x1 with 1x PBS. Add 500 µL pre-warmed iCLM media per well of a 24 well plate.
   5. Replace iCLM media every 2 - 3 days. Process plate 14 days after viral induction for immunocytochemistry (ICC) analysis of cardiac reprogramming.

3. Immunostaining of Reprogrammed MEFs

1. 14-days post-induction, carefully aspirate the media.
2. Rinse each well with 300 µL of ice-cold 1x PBS. Aspirate excess solution.
3. Fix cells with 250 µL of 4% paraformaldehyde (PFA) solution per well of a 24 well plate. Incubate 15 min at RT.
   NOTE: Fixed cells can be stored in PBS at 4 °C for 1 - 2 weeks before staining.
4. Permeabilize cells by washing wells x3 with 300 µL 0.1% PBS-TritonX 100 (PBST). Incubate 5 min at RT between washes. Aspirate excess solution after the last wash.
5. Block for 10 min at RT with 1x Universal Block Buffer at 300 µL/well.
6. Prepare (ICC) staining buffer: Add 1:1 of 1x PBS and 1x Universal blocking buffer. Dilute primary antibodies in ICC staining buffer and incubate antibodies overnight at 4 °C. Refer to the material section for recommended dilutions.
   1. Stain one pair of slides with mouse α-actinin, chicken αGFP, and rabbit Nppa for iPM and iAM identification.
   2. Stain one pair of slides with mouse α-actinin, chicken αGFP, and rabbit Myl2 for iPM and iVM identification.
7. The following day, wash wells x3 with 300 µL 0.1% PBST. Incubate 5 min at RT between washes. Aspirate excess solution after the last wash.
8. Prepare the secondary antibody dilutions in ICC staining buffer. Refer to the material section for recommended dilutions. Incubate secondary antibodies 1 h at RT, protected from light.
   1. Stain all slides with the following secondary antibodies: mouse Alexa-555, chicken Alexa-488, and rabbit Alexa-647.
9. Wash wells x3 with 300 µL 0.1% PBST. Incubate 5 min at RT between washes. Protect from light.
10. Add 2.4 µL of mounting media containing 1.5 µg/mL of 4',6-diamidino-2-phenylindole (DAPI) to a glass microscope slide. Carefully remove the coverslip from the well of the 24-well plate, remove excess solution, and transfer to the glass slide with mounting media. Gently press the coverslip to remove excess volume and air.
11. Seal slides with preferred nail polish or plastic sealant. Store mounted slides at 4 °C protected from light.

4. Identification of Cardiac Subtypes Using Confocal Microscopy

NOTE: For imaging, a confocal microscope equipped with at least 2 fluorescent detectors capable of spectral detection at 405, 488, 555, and 639 nm wavelengths is necessary in order to identify iPMs, iAMs, and iVMs. Image cells using a Plan-Apochromat 20X/0.75 objective or better. Using the manufacturer's image analysis software, scanning zoom images can achieve 40X-oil immersion quality images.

1. Image library: take 8-bit images with DAPI, Alexa-488, Alexa-555, and Alexa-647 channels (ch.). Pixel dwell time of 6 s, 1024 frame size, line step at 2, and averaging of 2 is sufficient for high resolution images.
2. For each slide, start from one edge and start scanning up and down in the red fluorescent channel (ch.) for α-actinin⁺ Sarcomere⁺ cells (Refer to Figure 3 and Figure 5A for examples). Sarcomere striations are easier to identify visually in the 555 nm wavelength.
   1. Once an α-actinin⁺ Sarcomere⁺ cell has been identified, switch to the green ch. and keep note if it is positive (iPM). Switch to the computer to assess the far-red 647 ch. (iAM or iVM).
      NOTE: Cells that are α-actinin⁺/Sarcomere⁺/Hnc4-GFP⁺/Nppa^-/Myl2^- are designated as iPMs. GFP expression will be seen throughout the cell (Figure 4A).
   2. Stain slides with α-actinin (mouse-Alexa555), Hcn4-GFP (chicken-Alexa488), Nppa (rabbit-Alexa647), and DAPI. Cells that are α-actinin⁺/Sarcomere⁺/Hnc4-GFP^-/Nppa⁺ are iAM. Nppa staining will appear perinuclear and punctate (Figure 4B).
   3. Stain slides with α-actinin (mouse-Alexa555), Hcn4-GFP (chicken-Alexa488), Myl2 (rabbit-Alexa647). Cells positive for α-actinin⁺/Sarcomere⁺/Hnc4-GFP^-/Myl2⁺ are iVMs. Myl2 staining will exhibit a striated form along the sarcomere filament. Due to variations in the quality of the staining and the Z-plane, striations may not always be easily visible (Figure 4C).

5. Quantification

NOTE: In order to assess the actual number of potentially reprogrammed MEFs, 2 wells of a 24-well plate are seeded in parallel to the experimental wells and are harvested one day after plating. The total number of cells plated is then determined by averaging the two wells. This becomes the actual total cells plated (aTotal).

1. Sarcomere⁺
   1. Visually inspect each cell on a coverslip for proper α-actinin⁺/Sarcomere⁺ (Right panels Figure 3) and record (Figure 5B-i).
   2. Tabulate the total number of α-actinin⁺/Sarcomere⁺ on each coverslip and divide by the actual total cells plated (aTotal) (Figure 5B-iii). For example, if aTotal = 12,500 cells, and 100 cells were α-Actinin⁺/Sarcomere⁺ then, 0.8% of the plated MEFs were reprogrammed. An average reprograming experiment will yield 1% α-Actinin⁺/Sarcomere⁺ cells (Figure 5C).
2. Subtype⁺
   NOTE: For the following steps, refer to Figure 5B/C for a representative iCLM quantification workflow. Briefly, for each sarcomere⁺ cell, tabulate if it is unique for either subtype (Figure 5B-i). Calculate % Subtype (Figure 5B-iii) by dividing the number of subtype⁺ cells over the average sarcomere⁺ cells x 100 (Figure 5B-i). To calculate the absolute % subtype efficiency (Figure 5B-iv), divide the subtype⁺ cell number from Figure 5B-i by the total number of cells seeded x 100 (Figure 5B-ii).
   1. For each of the α-actinin⁺/Sarcomere⁺ cells, assess if they are GFP⁺, Nppa⁺, or Myl2⁺.
   2. Tabulate total number of α-actinin⁺/Sarcomere⁺/Hnc4-GFP⁺/Nppa^-/Myl2^-, actinin⁺/Sarcomere⁺/Hnc4-GFP^-/Nppa⁺_, and α-actinin⁺/Sarcomere⁺/Hnc4-GFP^-/Myl2⁺ cells (Figure 5B-i)_.
   3. To calculate the percentage of each subtype, divide the number of Subtype⁺ cells by the total number of α-actinin⁺/Sarcomere⁺ cells in that well and multiply by 100. GHMT generates iPMs, iAMs, and iVMs at roughly equal ratios (Figure 5B-iii).
   4. To calculate the absolute number of subtype⁺ cells, divide the total number of subtype⁺ cells for the experimental condition by aTotal and multiply by 100 (Figure 5B-ii). On average iPMs represent 0.3% of the total infected cell population, iAMs 0.3%, and iVMs 0.25% (Figure 5B-iv).

Wyniki

Taking advantage of the PM-specific reporter mouse, we developed a multiplex immunostaining strategy to identify diverse endogenous myocytes as depicted in Figure 1. Following the reprogramming steps shown in Figure 2, induction of subtype-specific CMs can be detected as early as day 4²¹, albeit at a low-rate. By day 14, the experiment can be stopped and assessed for sarcomere organization (Figure 3) and subtype-sp...

Dyskusje

The present study provides a direct-reprogramming strategy for conversion of MEFs into a diverse set of cardiac subtypes via retrovirus-mediated expression of the cardiac transcription factors Gata4, Mef2c, Tbx5, and Hand2 (GHMT). Using a multiplex immunostaining approach in combination with a PM-specific reporter mouse, we are able to identify iAM, iVMs, and iPMs at single cell resolution. Such an assay allows for an experimental in vitro system capable of isolating the contributions of individual transcription...

Materiały

Name	Company	Catalog Number	Comments
DMEM	Sigma	D5796	Component of iCLM media, Plat-E media, fibroblast, and Transfection media
Medium 199	Thermo Fisher Scientific	11150059	Component of iCLM media
Fetal bovine serrum (FBS)	Sigma	F2442	Component of iCLM media, Plat-E media, fibroblast, and Transfection media
Insulin-Transferrin-Selenium G	Thermo Fisher Scientific	41400-045	Component of iCLM media
MEM vitamin solution	Thermo Fisher Scientific	11120-052	Component of iCLM media
MEM amino acids	Thermo Fisher Scientific	1601149	Component of iCLM media
Non-Essential amino acids	Thermo Fisher Scientific	11140-050	Component of iCLM media
Antibiotic-Antimycotics	Thermo Fisher Scientific	15240062	Component of iCLM media
B-27 supplement	Thermo Fisher Scientific	17504044	Component of iCLM media
Heat-Inactivated Horse Serum	Thermo Fisher Scientific	26050-088	Component of iCLM media
NaPyruvate	Thermo Fisher Scientific	11360-70	Component of iCLM media
Penicillin/Streptomycin	Thermo Fisher Scientific	1514022	Component of Plat-E media and fibroblast media
Puromycin	Thermo Fisher Scientific	A11139-03	Component of Plat-E media
Blasticidin	Gemini Bio-Products	400-128P	Component of Plat-E media
Glutamax	Thermo Fisher Scientific	35050-061	Component of Fibroblast media
Confocal laser scanning LSM700	Zeiss		For confocal analysis
FuGENE 6 transfection Reagent	Promega	E2692	Transfection reagent
Opti-MEM Reduced Serum Medium	Thermo Fisher Scientific	31985-070	Transfection reagent
Polybrene	Millipore	TR-1003-G	Induction reagent. Use at a final concentration of 8 μM/mL
Platinium-E (PE) Retroviral Packagin Cell Line, Ecotropic	CellBiolabs	RV-101	Retroviral pacaking cell line
Trypsin 0.25% EDTA	Thermo Fisher Scientific		For MEFs and Plat-E dissociation
Mouse anti α-Actinin (Clone EA-53)	Sigma	A7811	Antibody for confocal analysis. Use at 1:200
Chicken anti-GFP IgY	Thermo Fisher Scientific	A10262	Antibody for confocal analysis. Use at 1:200
Rabbit Pab anti-NPPA	Abgent	AP8534A	Antibody for confocal analysis. Use at 1:400
Rabbit Pab anti Myl2 IgG	ProteinTech	10906-1-AP	Antibody for confocal analysis. Use at 1:200
Vectashield solution with DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)	Vector Labs	H-1500	Dye for confocal analysis
Superfrost Plus Microscope slides	Thermo Fisher Scientific	12-550-15	25 x 75 x 1.0 mm
BioCoat Fibronectin 12 mm coverslips	NeuVitro Corp	GG-12-1.5	Coverslips for confocal analysis
100 μm cell strainer	Thermo Fisher Scientific	08-771-19
0.45 μm Syringes filters SFCA 25MM	Thermo Fisher Scientific	09-740-106	For virus filtration
6 mL Syringes	Covidien	8881516937	For virus filtration
Goat anti-Chicken IgY (H&L) A488	Abcam	AB150169	Secondary antibody for confocal analysis. Use at 1:400
Donkey anti-rabbit A647 IgG(H+L)	Thermo Fisher Scientific	A31573	Secondary antibody for confocal analysis. Use at 1:400
Goat anti-mouse IgG(H+L) A555	Thermo Fisher Scientific	A21422	Secondary antibody for confocal analysis. Use at 1:400
Triton X-100	Sigma	93443-100ml	For cell permeabilization
Dulbecco's PBS without CaCl2 and MgCl2 (D-PBS)	Sigma	D8537
Power Block 10x Universal Blocking reagent	Thermo Fisher Scientific	NC9495720	Dilute to 1x in H2O
16% Paraformaldehyde aqueous solution (PFA)	Electro Microscopy Sciences	15710	Use at 4% diluted in dH2O
6 cm  plates	Olympus	25-260
6-well plates	Genesee Scientific	25-105
24-well plates	Genesee Scientific	25-107
10 cm Tissue culture dishes	Corning	4239
15 cm  Tissue culture dishes	Thermo Fisher Scientific	5442
15 mL Conical tubes	Corning	4308
50 mL Conical tubes	Corning	4249
0.4% Trypan blue solution	Sigma	T8154	For viability
Ethyl Alcohol 200 proof	Thermo Fisher Scientific	7005
Bleach	Thermo Fisher Scientific	6009