Fractionation of embryonic cardiac progenitor cells and evaluation of their differentiation potential

Differentiation 105 (2019) 1–13

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Fractionation of embryonic cardiac progenitor cells and evaluation of their differentiation potential Tiam Feridooni, Kishore B.S. Pasumarthi

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Department of Pharmacology, Dalhousie University, Sir Charles Tupper Building, 5850 College Street, P.O. Box 15000, Halifax, Nova Scotia, Canada B3H 4R2

A B S T R A C T

Mid-gestation mouse ventricles (E11.5) contain a larger number of Nkx2.5+ cardiac progenitor cells (CPCs). The proliferation rates are consistently higher in CPCs compared to myocyte population of developing ventricles. Recent studies suggested that CPCs are an ideal donor cell type for replacing damaged tissue in diseased hearts. Thus, the ability to isolate and expand CPCs from embryos or stem cell cultures could be useful for cell fate studies and regenerative therapies. Since embryonic CPCs possess fewer mitochondria compared to cardiomyocytes, we reasoned that CPCs can be fractionated using a fluorescent mitochondrial membrane potential dye (TMRM) and these cells may retain cardiomyogenic potential even in the absence of cardiomyocytes (CMs). FACS sorting of TMRM stained embryonic ventricular cells indicated that over 99% of cells in TMRM high fraction stained positive for sarcomeric myosin (MF20) and all of them expressed Nkx2.5. Although majority of cells present in TMRM low fraction expressed Nkx2.5, very few cells (~1%) stained positive for MF20. Further culturing of TMRM low cells over a period of 48 h showed a progressive increase in MF20 positive cells. Additional analyses revealed that MF20 negative cells in TMRM low fraction do not express markers for endothelial cells (vWF, CD31) or smooth muscle cells (SM myosin). Treatment of TMRM low cells with known cardiogenic factors DMSO and dynorphin B significantly increased the percentage of MF20+ cells compared to untreated cultures. Collectively, these studies suggest that embryonic CPCs can be separated as a TMRM low fraction and their differentiation potential can be enhanced by exogenous addition of known cardiomyogenic factors.

1. Introduction The discovery of functional coupling between transplanted cardiomyocytes (CMs) and host myocardial cells (Roell et al., 2007; Rubart et al., 2003; Shiba et al., 2012) and the ability of transplanted embryonic CMs to confer protection against the induction of arrhythmia (Roell et al., 2007; Shiba et al., 2012) are significant steps towards the use of cell based therapies for myocardial repair. Several methods have been developed for differentiation of embryonic stem cells (ESCs) into cardiomyocytes (Mummery et al., 2003, 2012; Nemir et al., 2006; Yuasa et al., 2005). More recently, it has been shown that transplantation of human ES derived cardiac progenitor cells (CPCs) in end-stage heart failure patients can improve the function of akinetic and nonrevascularized areas with no complications (Menasche et al., 2015). While these new technologies have a high potential for regenerative therapies, current stem cell differentiation protocols are yet to yield a high degree of donor cell enrichment for cell based therapies (McMullen and Pasumarthi, 2007; Zhang and Pasumarthi, 2008). It is generally accepted that transplantation of undifferentiated stem cells can lead to formation of teratomas (Hentze et al., 2009; Nussbaum et al., 2007; Swijnenburg et al., 2005; Zhang et al., 2011). Thus, development of novel methods to isolate and expand pure populations of CPCs and CMs from embryos or stem cell cultures could be useful for

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regenerative therapies and cell fate studies. Although genetic selection methods are frequently used to enrich CPCs and CMs from stem cell cultures, they are not clinically feasible due to the prior requirement of genetic modification of cells using tedious techniques (Anderson et al., 2007; Gassanov et al., 2004; Hidaka et al., 2003; Huber et al., 2007; Pasumarthi and Field, 2002b). While cell surface marker based immunoselection approaches have been widely used to isolate CPCs from the postnatal hearts (Murry et al., 2004; Oh et al., 2003; Orlic et al., 2001), such approaches are not effective for the isolation of CPCs from embryonic hearts due to developmental differences in the expression profiles of these markers. Moreover, isolation of pure populations of CMs based on cell surface marker approach is challenging. Previous work from our laboratory established that embryonic CMs contain large number of mitochondria compared to undifferentiated cell population in E11.5 mouse ventricles using transmission electron microscopy (Zhang and Pasumarthi, 2007). Fukuda and colleagues reported that neonatal rat CMs have more mitochondria compared to other cell types and can be purified using the fluorescent mitochondria dye (tetramethylrhodamine methyl ester perchlorate; TMRM) (Hattori et al., 2010). Here, we reasoned that TMRM staining can be used to fractionate embryonic CPCs and CMs for cell fate studies and cell-based therapies. Similar to the findings from Fukuda's group (Hattori et al.,

Corresponding author. E-mail address: [email protected] (K.B.S. Pasumarthi).

https://doi.org/10.1016/j.diff.2018.11.001 Received 23 July 2018; Received in revised form 24 October 2018; Accepted 22 November 2018 Available online 23 November 2018 0301-4681/ © 2018 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

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Perchlorate), cells were incubated in 50 nM of TMRM solution (Molecular Probes Inc., 29851 Willow Creek Road, Eugene, OR USA, Cat#T668) diluted in Kreb's buffer (132 mM NaCl, 4 mM KCl, 1 mM CaCl2, 1.2 mM Na2HPO4, 1.4 mM MgCl2, 6 mM Glucose, 10 mM HEPES, pH 7.4) at 37 °C for 30 min, washed in PBS, centrifuged at 4000 rpm for 4 min and reconstituted in FACS-Buffer (4% BSA in PBS, w/v). Cells were then placed on ice and promptly used for FACS analysis (FACSAria, BD Biosciences, Franklin Lakes, New Jersey, USA). The sorted cells were centrifuged at 4000 rpm for 4 min and cultured for further analysis. The relative distribution of TMRM high and TMRM low cells for E11.5 and E17.5 ventricular cell preparations after FACS sorting were represented as percent cell distributions.

2010), results presented in this study demonstrated that CMs and nonCMs can be separated from the embryonic ventricular cell preparations into TMRM high and TMRM low cell fractions based on their mitochondrial content. Notably, we demonstrated that cells from the TMRM low fractions have the potential to differentiate into CMs even in the absence of any exogenous agents. Furthermore, cardiomyogenic differentiation in TMRM low cell fraction can be enhanced by the addition of recognized cardiomyogenic compounds. 2. Materials and methods 2.1. Animal maintenance and mouse strains All animal procedures were performed according to the Canadian Council on Animal Care guidelines and were approved by the Dalhousie University Committee on Laboratory Animal Care (Protocol# 14-013, 16–048). CD1 and C57/BL6 mice were obtained from Charles River Laboratories (Montreal, Canada). Generation of mice with Cre recombinase inserted into the Nkx2.5 allele was previously described (Stanley et al., 2002). The R26R-lacZ reporter strain was obtained from the Jackson Laboratories, (Bar Harbor, USA). All knock-in mouse lines were maintained in BL6 background and genotyping was performed as described in our previous studies (Feridooni et al., 2017; Hotchkiss et al., 2015; Zhang et al., 2015). Female mice were mated with males and E0.5 stage was designated as the noontime of the day when the copulation plug was found. Unless otherwise stated, CD1 mice were used for all experimental procedures.

2.4. Primary cell cultures and drug treatments TMRM high and TMRM low cell fractions were routinely plated on fibronectin (Sigma) coated 4-well chamber slides (Nunc, Rochester, New York, USA) at 1.2 × 105 density in each chamber. In some experiments, cells were cultured for 4, 24 and 48 h periods in 10% FBSDMEM and processed for immune cytochemistry. In other experiments, TMRM low cells were cultured for 4 h and subsequently treated for 48 h with various cardiomyogenic induction compounds including, 0.3% or 1% dimethyl sulfoxide (DMSO; Thermo Fisher Scientific, Nepean, Ontario, Canada), 100 nM dynorphin B (Sigma), 10 nM retinoic acid (Sigma), TMRM high conditioned media and 3 µM or 10 µM of 5-azacytidine (Sigma).

2.2. Isolation of primary embryonic ventricular cells and adult cardiac fibroblasts

2.5. Visualization of the mitochondrial content in TMRM high and low cell cultures

Timed pregnant female mice were anesthetized using 4% isoflurane and sacrificed by cervical dislocation. The embryos (E11.5 and E17.5) were removed from the uterus along with the placenta and placed in warm PBS supplemented with 1X antibiotic/antimycotic (Gibco, Burlington, Ontario, Canada). Next, whole hearts were extracted using a Leica MZ16SF stereomicroscope (Leica Microsystems, Richmond Hills, Ontario, Canada). The left and right ventricles were separated from the atria and major blood vessels, placed in 0.2% v/v type I collagenase (Worthington Biochemical Corp., Lakewood, New Jersey, USA) and rocked at 37 °C for 30 min (E11.5 ventricles) or 45 min (E17.5 ventricles) to digest the tissue. Following the incubation step, the tissue was triturated using a 200 µl pipette tip to dissociate cells from the remaining tissue pieces. Cells were then centrifuged at 4000 rpm for 4 min, and the cell pellet was neutralized with two subsequent washes of DMEM (Dulbecco's Modified Eagles Medium; Wisent, Saint Bruno, Quebec, Canada) containing 10% fetal bovine serum (FBS; Wisent). A hemocytometer was used to determine the total number of cells. Adult cardiac fibroblasts for control experiments were isolated from CD1 mouse hearts as described earlier (Gaspard et al., 2014).

FACS sorted TMRM high and low cell fractions were cultured for 24 h on fibronectin coated Delta T Dishes (Bioptechs Inc. Butler, PA, USA) at 1.0 × 105 cells/dish and stained with 50 nM TMRM (mitochondrial dye) and 30 nM calcein-AM (cell viability dye, Life Technologies Inc. 5250 Mainway, Burlington, ON, Canada) in Kreb's buffer for 30 min as described earlier. Images were collected using a Zeiss Cell Observer spinning disk confocal microscope (Carl Zeiss) and images were processed using the Ziess Zen lite software.

2.6. Immune cytochemistry Primary cultures were fixed in 1% paraformaldehyde solution (pH 7.4) for 3 min and permeabilized in 0.1% Triton X-100 (Sigma) for 4 min, followed by three consecutive 2 min PBS washes. Cells were then covered in blocking buffer solution [10% v/v goat serum (Gibco), 1% w/v bovine serum albumin (BSA; Thermo Fisher Scientific) in PBS]. After 1 h, blocking buffer solution was removed and replaced with blocking buffer containing primary antibodies of choice according to the concentrations listed in Table 1 for 1 h at room temperature. Next, the slides were washed with PBS three times for 3 min each and incubated with secondary goat anti-rabbit or goat anti-mouse antibodies conjugated to either Alexa Fluor 488 (1:200) or Alexa Fluor 555 (1:200) in blocking buffer for one hour. Cells were then washed three times and nuclei were counterstained by immersion in a solution of 1 µg/mL of Hoechst 33258 (Sigma) in PBS. Finally, the walls of the chamber slides were removed, and the slides were mounted with 0.1% propyl gallate (Sigma) solution [(0.1% w/v propyl gallate, 50% v/v glycerol (Thermo Fisher Scientific), 50% v/v PBS)]. Slides were then examined using the Leica DM2500 fluorescence microscope and images were captured using a Leica DFC 500 digital acquisition system. The number of cardiomyocytes (CMs; MF20+ cells) and nonmyocytes (NMCs; MF20- cells) were quantified and represented as number of cells per millimeter squared.

2.3. TMRM staining and fluorescence activated cell sorting (FACS) Embryonic ventricular cells or adult cardiac fibroblasts were isolated and neutralized with 10% FBS-DMEM as described above. To ensure that single cell suspension was obtained, cells were passed through a 40 µM mesh filter, and centrifuged at 4000 rpm for 4 min. In experiments in which E11.5 ventricular myocytes were used, generally 6–8 pregnant mice were sacrificed and approximately 10 embryonic hearts were obtained per pregnancy resulting in ~4.5 × 106 cells prior to preparations for TMRM staining and FACS analysis. In experiments in which E17.5 ventricular myocytes were used, generally 1–2 pregnant mice were sacrificed and approximately 10 embryonic hearts were obtained per pregnancy resulting in ~5.0 × 106 cells prior to FACS analysis. For TMRM staining (Tetramethyl rhodamine Methyl Ester 2

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E11.5 ventricular cells indicated that the number of CMs is significantly higher in the TMRM high subpopulation (97.3 ± 9.9 cells/mm2) compared to that of the TMRM low subpopulation (1.2 ± 0.3 cells/ mm2) (Fig. 3 G). Inversely, the number of NMCs was significantly lower in the TMRM high subpopulation (1.0 ± 0.5 cells/mm2) compared to the TMRM low subpopulation (48.5 ± 2.0 cells/mm2) (Fig. 3 H). Immunolabelling of fractionated TMRM high and low E17.5 ventricular cells indicated that the number of CMs is significantly higher in the TMRM high subpopulation (201.1 ± 17.9 cells/mm2) compared to the TMRM low subpopulation (1.2 ± 0.5 cells/mm2) (Fig. 3 I). Inversely, the number of E17.5 NMCs was significantly lower in the TMRM high subpopulation (0.6 ± 0.3 cells/mm2) compared to the TMRM low subpopulation (41.2 ± 8.5 cells/mm2) (Fig. 3 J).

Table 1 Primary antibodies and dilutions used for immunofluorescence experiments. Primary Antibody

Dilution

Source / Catalogue #

α-Smooth muscle actin

1:50

β-galactosidase

1:100

Cluster of differentiation 31

1:50

Connexin 40

1:100

HCN4

1:100

Nestin

1:100

Sarcomeric myosin (MF20)

1:100

Von Willebrand Factor

1:50

Sigma Aldrich Catalogue #: A5228 Cappel ICN Catalogue #: 55976 Developmental Studies Hybridoma Catalogue #: 2H8-c Alpha Diagnostic Catalogue #: Cx40-A Alomone Laboratories Catalogue #: APC−052 Developmental Studies Hybridoma Catalogue #: Rat−401 Developmental Studies Hybridoma Catalogue #: MF−20 Santa Cruz Biotechnology Catalogue #: sc−14014

3.2. Differentiation potential of TMRM low subpopulation into mature cardiomyocytes in vitro Since results from the previous section indicated very low number of differentiated CMs (MF20+) in the FACS sorted E11.5 and E17.5 TMRM low subpopulations 4h post culturing, we next examined the differentiation capacity of MF20- cells into MF20+ cells over time. To do so, E11.5 and E17.5 ventricular cells were FACS sorted according to their mitochondrial content and the TMRM low subpopulations were cultured for 4, 24 and 48 h. Immunostaining of the TMRM low E11.5 ventricular cells revealed a very low number of MF20+ 4 h post culturing (Fig. 4 A). In contrast, the density of MF20+ cells in cultured TMRM low subpopulation progressively increased after 24–48 h (Fig. 4 A-C) of culture period. Quantitative analysis revealed that compared to 4 h (1.2 ± 0.3 cells/mm2), the number of MF20+ CMs significantly increased by 24 h (15.6 ± 1.4 cells/mm2) and 48h (29.6 ± 2.3 cells/ mm2) (Fig. 4 D). Quantification of MF20 negative cells indicated that compared to 4h (48.5 ± 2.0 cells/mm2), the number of NMCs significantly increased 24 h after culturing (88.0 ± 8.3 cells/mm2) and 48 h (180.4 ± 17.8 cells/mm2) post culturing (Fig. 4 E). FACS sorted E11.5 TMRM low ventricular cells cultured for 72 h indicated a significant increase in MF20+ cells (93.8 ± 13.3 cells/mm2) compared to 4, 24, and 48 h, however, the culture was overtaken with an overwhelming number of MF20- cells (1071.0 ± 139.0 cells/mm2) (data not shown). Thus, the 48 h time point was used for further studies. Immunolabelling of TMRM low E17.5 ventricular cell cultures indicated that compared to 4 h cultures (1.2 ± 0.6 cells/mm2), the number of CMs significantly increased after 24 h (11.5 ± 2.3 cells/ mm2) and 48 h culture periods (37.4 ± 4.8 cells/mm2) (Fig. 4 F). Quantification of MF20- cells indicated that compared to the 4 h cultures (41.2 ± 8.5 cells/mm2), the number of NMCs significantly increased in 24 h (181.4 ± 16.1 cells/mm2) and 48 h cultures (150.5 ± 8.6 cells/mm2) (Fig. 4 G) in E17.5 TMRM low cultures. Collectively, these data suggest that TMRM low subpopulation contains CPCs capable of generating cardiomyocytes at both E11.5 and E17.5 stages of ventricular development. TMRM low cells from both E11.5 and E17.5 stages appear to possess a similar magnitude of cardiomyogenic potential (E11.5: 29.6 ± 2.3 cells/mm2 Vs. E17.5: 37.4 ± 4.8 cells/mm2 at 48 h time points). Furthermore, NMC cells appear to expand exponentially in TMRM low cultures from both developmental stages along with the newly formed cardiomyocytes.

2.7. Statistical analysis Statistical analysis was performed using the Graphpad Prism Version 5.01 (Graphpad Software, San Diego, USA). Data are presented as mean ± standard error of the mean (SEM). Multiple group comparisons were analyzed by ANOVA and Tukey multiple comparison post hoc test. A two-tailed unpaired t-test was used to compare between two groups. Significance for all analyses was assigned at P < 0.05. For each experiment, the number of experiments/replicates is represented in the corresponding figure legends. 3. Results 3.1. Fractionation of CPCs and CMs from a mixed population of E11.5 and E17.5 embryonic ventricular cells based on mitochondrial content E11.5 or E17.5 ventricular cells dispersed from the CD1 embryonic ventricles were stained with TMRM and subjected to FACS sorting analysis (Fig. 1 A, B and D, E). As additional controls, unstained E11.5 ventricular cells as well as adult cardiac fibroblasts stained with or without TMRM were also processed for FACS analysis (Fig. 1 C, F and G-J). Scatter plots collected from the FACS analysis revealed three distinct cell populations in the TMRM stained E11.5 and E17.5 ventricular cells compared to unstained cell preparations (Fig. 1A-J). Unstained cell preparations exhibited only background fluorescence in the TMRM channel. Notably, TMRM high and TMRM low fractions (fractions 1 and 2 respectively) from E11.5 and E17.5 ventricular cells could be readily cultured in vitro. In contrast, TMRM negative cells (fraction 3) contained dead cells and debris and thus could not be cultured. Quantitative analysis revealed that TMRM stained E11.5 ventricular cells consisted of 27.0 ± 1.5% TMRM high and 73.0 ± 1.5% TMRM low cell populations (Fig. 2A). In contrast, TMRM stained E17.5 ventricular cells consisted of 57.0 ± 0.6% TMRM high and 42.0 ± 0.6 TMRM low cell populations (Fig. 2B). To further validate our FACS findings regarding the mitochondrial content of TMRM high and low fractions, FACS sorted E11.5 ventricular cells were cultured and stained with TMRM and a cell viability marker calcein green. Using confocal imaging, it was clear that the cells in TMRM high fraction contain more mitochondrial content (Fig. 2 C-E) compared to the cells in TMRM low fraction (Fig. 2 F-H). To determine the CM and non-CM cell (NMC) distribution within each subpopulation of TMRM positive cells, FACS sorted cells were cultured on fibronectin coated chamber slides for 4 h, fixed and processed for immunolabelling with differentiation marker sarcomeric myosin heavy chain (MF20). Cells positive for MF20 were noted as CMs (MF20+) and those negative for MF20 were noted as NMCs (MF20-) (Fig. 3 A-F). Further quantification of fractionated TMRM high and low

3.3. In vitro survival and expansion of TMRM high cardiomyocyte cultures To determine if there are any differences in viability and expansion characteristics of pure CM populations between E11.5 and E17.5 stages, FACS sorted TMRM-high cells were plated on fibronectin coated chamber slides and maintained for 4, 24 or 48 h culture periods (Fig. 4 H-J). Immunolabelling of E11.5 TMRM high cultures with MF20 antibodies indicated that compared to 4h (97.3 ± 9.9 cells/mm2), the number of MF20+ CMs did not significantly increase by 24 h (107.2 ± 21.5 cells/mm2) or 48 h (155.6 ± 3.2 cells/mm2) time 3

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Fig. 1. Representative FACS experiments for the mitochondrial content marker TMRM in unfixed and unpermeabilized embryonic ventricular cells and adult cardiac fibroblasts. Histograms in the upper panels represent cell numbers and the corresponding TMRM fluorescence intensity. Lower panels are dot plots indicating measured fluorescence of individual cells from the representative FACS experiments. Results from the unstained cell preparations or negative control (Neg) experiments (C, F and H, J) were used to set the threshold for the levels of TMRM intensity in panels A, B D, E, G and I. Compared to the unstained E11.5 ventricular cells (C), a shift to the right in the TMRM staining pattern was evident in stained E11.5 (A) and E17.5 (B) ventricular cell preparations. In the dot plots of TMRM stained embryonic ventricular cells (D, E), three distinct fractions were visible, TMRM high (F1), TMRM low (F2) and TMRM negative (F3). Adult cardiac fibroblasts (CF) were also used to test the assay's capability to sort cells according to their mitochondrial content (G, H and I, J). Compared to the unstained adult CF (H), a shift to the right in the staining pattern also occurred in CF preparations stained with TMRM (G). In addition, the majority of adult CF stained were either TMRM low (F2) or TMRM negative with no cells in TMRM high region (F1; see panel I).

points (Fig. 4 K). Quantification of MF20- cells (NMC) indicated that compared to 4h (0.96 ± 0.5 cells/mm2), the number of NMCs did not significantly increase at 24 h (4.1 ± 2.4 cells/mm2) and 48 h (7.3 ± 2.4 cells/mm2) post culturing (Fig. 4 L). Immunolabelling of E17.5 TMRM-high cells with MF20 antibodies indicated that compared to 4 h (201.1 ± 17.8 cells/mm2), the number of CMs did not significantly increase after 24 h (205.6 ± 13.6 cells/ mm2) and 48 h (248.0 ± 32.7 cells/mm2) post culturing (Fig. 4 M). Quantification of MF20 negative cells indicated that compared to 4h (0.6 ± 0.3 cells/mm2), there was no significant increase in the number of NMCs 24 h (0.2 ± 0.2 cells/mm2) and 48 h (0.25 ± 0.25 cells/ mm2) after culturing (Fig. 4 N). In contrast to TMRM low cultures, TMRM high cultures could not be accurately analyzed for CM and NMC content in some fields at 72 h time points due to overcrowding of CMs. These results indicate that TMRM-high cultures from both E11.5 and E17.5 ventricles can be maintained as pure CM populations (> 98%) with a minimal expansion of NMC over 48–72 h culture periods.

percentage (~20%) of TMRM low (Fig. 5 A-C) and TMRM high (Fig. 5 D-F) subpopulations cultured for 48 h. In E11.5 TMRM low cells, HCN4 was expressed in both CMs (MF20+) and NMCs (MF20-) cells (Fig. 5C) 48 h post culturing. Cx40 is a member of the gap junction channels, which is responsible for coupling myocytes involved in mediating conduction in the heart. In the mouse heart, Cx40 expression is restricted to atrial and ventricular conductive myocytes (Miquerol et al., 2003). Cx40 was also expressed in a small percentage (~5%) of cells in TMRM low Fig. 5 (G-I) and TMRM high (Fig. 5 J-L) subpopulations.

3.5. Further characterization of NMCs from E11.5 TMRM labeled cell fractions Our next goal was to characterize the NMCs of E11.5 TMRM low fraction. To determine if there are any endothelial cells within the 48 h TMRM low cell cultures, we used the endothelial markers von Willebrand Factor (vWF) and CD31. Notably, E11.5 TMRM low cultures were negative for vWF (Fig. 6 A, D), yet, a small percentage (~0.1%) of cells with positive staining for CD31 were seen (Fig. 6 B, E). However, immunolabelling with antibodies for α-smooth muscle actin (α-SMA) did not reveal the presence of any smooth muscle cells within TMRM low cultures (Fig. 6 C, F). Although nestin has generally been suggested as a neural stem cell marker (Rietze et al., 2001), its expression has been identified in mid-embryonic period of developing mouse heart (Calderone, 2012). Consistent with previous findings, immunolabelling of TMRM low cultures revealed a prominent nestin staining in majority of cells (Fig. 6 G, J). In previous studies, we showed that both CPC and CM cell types express the transcription factor Nkx2.5 using transmission electron microscopy (Zhang and Pasumarthi, 2007) as well as by using a Nkx2.5Cre and ROSA-LacZ (NCRL) based lineage tracking mouse reporter system (Feridooni et al., 2017; Hotchkiss et al., 2015; Zhang et al.,

3.4. Ventricular conduction system markers are expressed in a small number of cells in both TMRM high and TMRM low cell cultures Ventricular conduction system (VCS) patterning is yet to develop at E11.5 stage and VCS cells can be identified by the presence of few myofilament bundles (Viragh and Challice, 1977). We next sought to determine whether the TMRM high and low cell fractions also express VCS markers after culturing for 48 h. To this end, cultured TMRM cell fractions were immunolabelled with VCS specific markers HCN4 and Cx40. HCN4 is a member of potassium/sodium hyperpolarization activated cyclic nucleotide-gated channels and is predominantly expressed in both sinus nodal cells (Schulze-Bahr et al., 2003) and the Purkinje fibers of VCS (Han et al., 2002) in the mammalian heart. Our immunostaining results revealed that HCN4 was expressed in a small 4

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Fig. 2. Percent distribution and representative images of FACS sorted TMRM high and low cells from the E11.5 and E17.5 embryonic ventricular cell preparations. (A, B) Quantification of TMRM high and TMRM low cell numbers in (A) E11.5 and (B) E17.5 ventricular cell preparations. * p < 0.005 Vs. TMRM high fraction, unpaired Student's t-test. Results are mean ± SEM of 4 independent experiments/group. (C-H) Representative images of (C-E) TMRM high and (F-H) TMRM low fractions stained with calcein green and TMRM (red). Left panels indicate cells stained with calcein, green, center panels show the same field of cells stained with TMRM and the right panels represent merged images. Scale bars = 10 µm (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

2015). It has also been shown that cardiac fibroblasts are void of Nkx2.5 expression (Kasahara et al., 1998). To further examine whether the NMCs of E11.5 TMRM fractions also express Nkx2.5, the double knock-in NCRL mouse model approach was used as previously described. The NCRL reporter approach was used instead of conventional

Nkx2.5 immunostaining due to the fact that Nkx2.5 promoter activity is subjected to spatiotemporal regulation in the embryonic ventricles (Searcy et al., 1998). In this regard, NCRL mice facilitate an efficient Cre-loxP based tracking of Nkx2.5+ progenitors and their descendants independent of the developmental regulation of Nkx2.5 promoter

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Fig. 3. Characterization of FACS sorted E11.5 and E17.5 ventricular cells stained with the TMRM. (A-F) Representative images of TMRM high (A-C) and TMRM low (D-F) cells stained with the sarcomeric myosin antibodies (MF20, A, D), Hoechst nuclear stain (B, E), and the last column shows merged images (C, F). Scale bars = 100 µm. (G-J) Quantification of the number of cardiomyocytes (CMs, G and I) and nonmyocytes (NMCs, H and J) in TMRM sorted fractions of E11.5 (G, H) and E17.5 (I, J) ventricular cells. Data is presented as the number of cells per square millimeters (mm2). * p < 0.05 Vs. TMRM low fraction, unpaired Student's t-test. Results are mean ± SEM of 4 independent experiments/group.

activity. Since β-galactosidase (β-gal) staining alone is not enough to confirm the myogenic differentiation status of a cell lineage, simultaneous labeling of cells with antibodies specific for β-gal and sarcomeric myosin was used to identify NMC/CPC (β-gal+/MF20-) and cardiomyocytes (β-gal+/ MF20+). E11.5 NCRL embryonic ventricular heart cells were sorted based on their mitochondrial content and cultured for 48 h. Double immunolabelling of the NCRL cells from the TMRM low fraction for β-gal (Nakajima et al., 2006) and MF20 indicated a strong expression of β-gal in both MF20- (NMCs) and MF20+ (CMs) cells (Fig. 6 H, I and K, L). Collectively, these results indicate that NMCs from TMRM low cultures harbor Nkx2.5+ CPCs that can give rise to working and conduction system myocytes but do not differentiate into other cell lineages under routine culture conditions. However, the presence of CD31 and nestin

positive cells also suggests that TMRM low cells may harbor a small percentage of multi-potent progenitor cells, which may differentiate into other cell lineages under specialized culture conditions.

3.6. Exogenous treatment of E11.5 TMRM low cultures with cardiomyogenic induction factors enhance cardiomyocyte formation in vitro We examined the effects of high and low concentrations of DMSO, dynorphin B, retinoic acid and high and low concentrations of 5-azacytidine on differentiation of E11.5 TMRM low cell cultures. Concentrations of exogenous factors were chosen based on the published literature. We also examined the effects of TMRM high cell culture conditioned media on differentiation of TMRM low subpopulation as CMs in TMRM high cultures may release factors that could contribute 6

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Fig. 4. Cardiomyogenic differentiation potential of FACS sorted TMRM low and TMRM high fraction cells in the presence of serum. TMRM low (A-C) and TMRM high (H-J) fraction cells from E11.5 and E17.5 ventricles were cultured for 4, 24 and 48 h and immunolabelled with sarcomeric myosin antibodies (MF20, red) and Hoechst nuclear stain (blue). Scale bars = 100 µm. Representative images obtained from the random fields were used to quantify the number of cardiomyocytes (CMs; MF20+) and nonmyocytes (NMCs; MF20-) per square millimeter area (mm2) in TMRM low (D-G) and TMRM high (K-N) fractions from E11.5 (D, E, K, L) and E17.5 (F, G, M, N) ventricular cells. * p < 0.05 Vs. 4 h culture, #p < 0.05 Vs. 24 h cultures, one-way ANOVA with Tukey's multiple comparison test. Results are mean ± SEM of 4 independent experiments/group (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

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Fig. 5. Subcellular localization of conduction system markers, HCN4 and Cx40 in TMRM low and high fractions of FACS sorted E11.5 ventricular cells. FACS sorted TMRM low (A-C; G-I) and TMRM high (D-F; J-L) cells were cultured for 48 h and immunolabelled with antibodies specific for sarcomeric myosin (MF20, red), K+/ Na+ hyperpolarization-activated cyclic nucleotide-gated channel (HCN4; green) or connexin 40 antibodies (Cx40; green) and Hoechst nuclear stain (blue). (A-C) In the TMRM low fraction, HCN4 labeling in both cardiomyocytes (CMs, long arrow) and nonmyocytes (NMCs, arrow head) localized mainly in the cytoplasm. (D-F) Similarly, in the TMRM high fraction HCN4 labeling was strong in CMs (long arrow) and localized mainly in the cytoplasm. (G-I) In the TMRM low fractions, Cx40 labeling was only visible in the CMs (long arrow) localized mainly in the membrane of adjacent cells. (J-L) Similarly, in the TMRM high fractions, Cx40 labeling was strong in the CMs (long arrow) localized mainly in the membrane of adjacent cells. Scale bars = 50 µm (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

compared to the untreated group (30.9 ± 2.8 cells/mm2), the number of MF20+ cells significantly increased in cultures treated with 1% DMSO (68.8 ± 8.6 cells/mm2), 0.3% DMSO (83.0 ± 10.6 cells/mm2) and 100 nM dynorphin B (76.5 ± 5.9 cells/mm2) (Fig. 7 E). In contrast, there were significant decreases in the number of MF20+ cells in cultures treated with 10 nM retinoic acid (17.4 ± 2.2 cells/mm2), conditioned media of TMRM high subpopulation (19.9 ± 2.6 cells/

to the differentiation of CPCs. To examine the effects of various factors, TMRM low cells were cultured for 48h in the presence or absence of exogenous factors (Fig. 7 A-D). Subsequently, cells were immunolabelled with MF20 antibodies and the CM number was quantified and expressed as number of MF20+ cells per squared millimeter using previously reported image analysis methods (Gaspard and Pasumarthi, 2008). Quantification of the MF20+ cells (CMs) indicated that 8

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Fig. 6. Further characterization of FACS sorted TMRM low fractions from E11.5 ventricular cells after a 48-h culture period. (A-F and G, J) Wild type E11.5 TMRM low cells were cultured for 48 h and then immunolabelled to characterize cell types present. Micrographs represent paired images of cells from the same field stained with the antibody of interest and Hoechst nuclear stain (Nuc) as indicated by the labels in figure panels. Cells were stained with (A, D) von Willebrand factor (vWF), (B, E) cluster of differentiation 31 (CD31), (C, F) α-smooth muscle actin (α-SMA) and (G, J) nestin to distinguish the phenotype of the nonmyocytes (NMCs) seen in TMRM low cultures. (H, I, K, L) E11.5 TMRM low cells were FACS sorted from double knockin embryos (Nkx2.5 Cre X Rosa-lacZ) and cultured for 48 h in order to genetically label Nkx2.5+ lineage by the expression of β-galactosidase (β-Gal). Cells from the same field were co-stained with antibodies for β-Gal (red), sarcomeric myosin (MF20; green) and Hoechst nuclear stain (blue; Nuc) to distinguish between cardiomyocytes (CMs; MF20+) and nonmyocytes (NMCs; MF20-). Immunolabelling for β-Gal was prominent in the majority of the TMRM low cells indicating an Nkx2.5+ lineage. Scale bars: 100 µm for A-F and G, J and 50 µm for H, I, K, L panels (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

mm2), 3 μM 5-azacytidine (14.7 ± 1.6 cells/mm2) and 10 μM 5-azacytidine (11.1 ± 1.0 cells/mm2) compared to DMSO treated groups or dynorphin B treated group but not compared to the untreated group (Fig. 7E). These results suggest that additional factors such as dynorphin B or DMSO are necessary to increase cardiomyogenic induction in

TMRM low cultures in vitro. 4. Discussion Lineage tracking studies revealed that the Nkx2.5+ cells in the 9

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Fig. 7. Differentiation potential of TMRM low ventricular cells in response to cardiogenic compounds. (A-D) E11.5 TMRM low cells were cultured for 48h in the presence or absence of various cardiomyogenic factors and immunolabelled with sarcomeric myosin antibodies (MF20; red) and Hoechst nuclear staining (blue). Cardiomyocytes are indicated by arrows and nonmyocytes are indicated by arrowheads. Scale bars = 100 µm. E) Quantification of the number of cardiomyocytes (CM) in different culture conditions per square millimeter area. * p < 0.005 Vs. untreated cultures, #p < 0.005 Vs. DMSO or Dynorphin B treated cultures but not untreated cultures, one-way ANOVA with Tukey's multiple comparison test. Results are mean ± SEM of 4 experiments/group (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

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Ventura et al., 2003a, 2003b). It is possible that dynorphin B may exert similar effects in TMRM low cell cultures to induce CM differentiation. Treatment of TMRM low fraction with retinoic acid (RA) resulted in a significant decrease in the number of CMs. While previous studies suggested that RA plays an essential role in the differentiation of ventricular cell phenotype and proper development of ventricular trabeculae (Lin et al., 2010; Niederreither et al., 2001), RA treatment was also shown to limit FGF signaling and regulate the size of the cardiac field (Keegan et al., 2005; Ryckebusch et al., 2008). In contrast to our findings, RA at 10 nM concentration was shown to increase the number of CMs with Purkinje- and ventricular-like phenotype in ES cell cultures (Wobus et al., 1997). This difference can be attributed to the timesensitive and dose dependent roles of RA in differentiation of CPCs into CMs. It was shown that treatment of ES cell cultures at an early stage with RA at 100–500 nM concentrations resulted in preferential atrial cell-like phenotype, whereas treatment at later stages did not affect cardiogenic differentiation (Gassanov et al., 2008; Lee et al., 2017). Additionally, RA when used at 50 nM concentration was shown to increase the expression of extracellular matrix genes (type IV collagen and laminin B1 during differentiation of F9 embryonal carcinoma cells into visceral endoderm (Rogers et al., 1990)). Absence of cardiomyogenic induction in TMRM low cultures treated with 10 nM RA in this study could be due to reductions in the expression of CM differentiation markers and/or increases in extracellular matrix markers as observed with F9 embryonal carcinoma cells (Rogers et al., 1990). Additional studies with multiple doses of RA are required to confirm whether RA plays any role in the differentiation of embryonic CPCs present in the mid to late gestation ventricles. Furthermore, several studies have highlighted the cardiomyogenic potential of 5-azacytidine (5-Aza) in ESCs and P19 EC cells (Abbey and Seshagiri, 2013; Lopez-Ruiz et al., 2014) as well as in mesenchymal stem cell cultures (Supokawej et al., 2013) and adult Sca1+ CPC cultures (Oh et al., 2003). However, both low and high concentrations of 5-Aza did not induce cardiomyocyte differentiation in TMRM low fractions. Interestingly, exogenous addition of other cardiomyogenic factors such as cardiotropin-1 and oxytocin in combination with 5-Aza enhanced CM induction in P19 EC cells (Paquin et al., 2002; Xinyun et al., 2010). Thus, co-administration of exogenous growth and cardiomyogenic factors in combination with 5-Aza may be necessary to induce further CM differentiation in TMRM low fraction. Although conditioned media from TMRM high cells did not have any effect on the degree of cardiomyogenic induction compared to untreated cultures, secretion of cardiomyogenic induction factors from CMs cannot be ruled out since these factors may be labile under the conditions used in this study. In future studies, it is also important to examine the ability of these cardiomyogenic factors on induction of other cell lineages such as smooth muscle cells or endothelial cells in TMRM low cultures. It has been shown that mid-gestation ventricular cells can form larger intracardiac grafts compared to cells from late-gestation ventricles in cell transplantation experiments (Zhang et al., 2015). However, it is not clear whether the high engraftment efficiency of midgestation ventricular cells is mainly due to high CPC content or due to higher cell cycle kinetics of both CPCs and CMs at that stage compared to later development stages (Zhang et al., 2015). Given the high purity of CM population in TMRM high fractions, it should be feasible to compare the engraftment efficiencies of TMRM high fractions from different stages of ventricular development in future experiments. Similarly, engraftment efficiencies of TMRM low and high fractions for a given developmental stage can also be compared in future experiments. Further, TMRM low cell fractions from developing ventricles can also serve as a model system for studying the effects of new compounds on differentiation of CPCs into CMs and other cardiac-specific cell types. It is also important to determine the beta-AR responses and calcium handling properties of embryonic CPC and CM populations in future studies.

developing heart are multipotent in nature and are able to give rise to cardiac, smooth muscle, endothelial, and conduction system cells (McMullen et al., 2009; Moretti et al., 2006; Wu et al., 2006). Notably, embryonic ventricles were shown to contain a large number of CPCs which can give rise to working CMs and conduction system cells in primary culture studies (Govindapillai et al., 2018; McMullen et al., 2009). In agreement with these studies, we demonstrated that majority of cells from the TMRM low fraction are derived from the Nkx2.5+ cell lineage. Furthermore, characterization of TMRM fractions using VCS markers, HCN4 and Cx40 (Govindapillai et al., 2018), provided evidence that TMRM low fraction cells possess the ability to differentiate into VCS cells. Notably, TMRM low fractions from E11.5 ventricles did not express markers for smooth muscle and endothelial cells under routine culture conditions. Although we have not further characterized the TMRM low cell fractions from E17.5 ventricles, some of these cells are likely to express markers for endothelial cells, smooth muscle cells and fibroblasts. Indeed, all three cell types were shown to be differentiated from distinct sets of progenitor cells in later stages of heart development (Ieda et al., 2009; Wu et al., 2012). The presence of nestin positive undifferentiated cells in E11.5 TMRM low cell fraction suggests that these cells may differentiate into other cell lineages. Nestin, an intermediate filament protein, is known to be expressed in a variety of cell types including neural progenitor cells, endothelial progenitor cells and cancer stem cells (Matsuda et al., 2013; Neradil and Veselska, 2015; Rietze et al., 2001). Although our studies did not provide any evidence for a robust differentiation of E11.5 TMRM low cells into vascular cell types, it is possible that some of these cells may differentiate into vascular smooth muscle cells and endothelial cells under specialized culture conditions. This notion is consistent with a suggested angiogenic role for nestin in a number of tumor models (Matsuda et al., 2013). While this protein is considered as a good biomarker for a variety of cancer cell types (Tampaki et al., 2014), intracardiac or hind leg transplantation of nestin positive embryonic ventricular cells did not lead to formation of teratomas in short-term studies (Feridooni et al., 2017; Hotchkiss et al., 2014; Zhang et al., 2015). It is unlikely that these nestin positive TMRM low cells will form teratomas in long-term studies due to cessation of cell cycle activity in ventricular cells during later stages of development (Pasumarthi and Field, 2002a; Zhang et al., 2015). We observed significant increases in CM numbers (15–30 fold) in untreated TMRM low fractions after culturing them for 24–48 h when compared to 4 h cultures. Although these increases could be attributed to a high degree of embryonic CM proliferation (Hotchkiss et al., 2012; Zhang et al., 2015), it is unlikely that TMRM low cell derived CMs can undergo 4–5 cell divisions within 24–48 h period to account for changes in CM numbers. This notion is further supported by the observations that CMs present in TMRM high fractions do not expand significantly over time in culture. Moreover, previous studies revealed that embryonic CPCs (Nkx2.5+ANF-MLC2V-MF20-) can differentiate into MF20+ CMs using real-time imaging techniques (McMullen et al., 2009; Zhang et al., 2015). Collectively, these findings suggest that increases in the number of CMs in TMRM low cultures result from the differentiation of embryonic CPCs present in those fractions. Previous studies revealed that addition of certain factors to stem cell cultures can increase the magnitude of cardiomyogenic differentiation. For instance, DMSO and dynorphin B have been shown to induce CM differentiation and trigger expression of GATA4 and Nkx2.5 genes, appearance of α-myosin heavy chain and myosin light chain-2V in murine P19 embryonal carcinoma cells (Gong et al., 2013; Ventura and Maioli, 2000; Ventura et al., 2003b). Consistent with these earlier reports, treatment of TMRM low fraction cell cultures with DMSO or dynorphin B significantly increased cardiomyocyte differentiation. Cardiomyogenic induction mediated by dynorphin B in P19 cells has been attributed to its ability to activate subcellular and nuclear isoenzymes of protein kinase C (PKC) resulting in increased expression of cardiomyogenic genes GATA4 and Nkx2.5 (Ventura and Maioli, 2000; 11

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Acknowledgements

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