**1. Introduction**

Mesenchymal stromal cells (MSC) represent a multipotent cell population capable to differentiate into different cell types [1]. They are an easily-accessible cell source as they can be isolated at high yields from various kinds of human tissue, such as umbilical cord, bone marrow, dental pulp, adipose tissue, placenta, etc. [1]. The common mesenchymal cell types that emanate from MSC are osteocytes, chondrocytes, and adipocytes [2]. Due to their plasticity, MSC are considered as one of the most important cell types for the application in regenerative medicine as demonstrated by a huge number of pre-clinical studies and several clinical trials [3,4]. In addition, MSC mediate immunomodulatory and immunosuppressive effects that promote wound healing and tissue repair, while showing no teratoma formation post transplantation [5]. Nowadays, it is commonly accepted that the observed therapeutic impact induced by MSCs is mainly based on the secretion of paracrine factors rather than on the differentiation into cardiomyocytes.

In recent years, MSC have also been utilized for the generation of mesenchymal as well as non-mesenchymal cell lineages, including neuron-like, hepatocyte-like, and cardiac-like cells [6–10]. Despite these promising results, the differentiation of human MSC into fully mature cardiomyocytes bearing all their respective phenotypical and functional characteristics is difficult [11–15]. As MSC are located in various tissues, they represent a heterogeneous progenitor cell population dependent on the tissue source and the individual donor [16]. This heterogeneity could explain the variety in differentiation characteristics [17–19]. Therefore, it remains to be investigated which type of MSC favorably undergoes cardiac trans-differentiation, thus, is a suitable candidate for cardiac reprogramming strategies. Detailed knowledge about the cardiac differentiation potential of specific MSC populations is even more important as some studies showed enhanced therapeutic effects following cardiovascular lineage commitment of MSC [12].

The development of an approach to efficiently control the cardiac differentiation of MSC would be a crucial step for the production of patient-derived cardiomyocytes without any ethical concerns. As such, they can also serve as a model system, beneficial for basic cardiovascular research, drug screening, and translational applications. Currently, several re/programming strategies exist to guide the mesenchymal and non-mesenchymal differentiation of MSC, such as treatment with small molecules and cytokines, exposure to metabolic stress, co-culture experiments, or overexpression of regulatory proteins [20–24]. For the potential clinical use, transient, non-integrative reprogramming approaches are preferred to prevent permanent alterations of the genome and to reduce tumorigenic risk. Small non-coding RNAs, like microRNAs (miRNA) and chemically modified messenger RNA (mRNAs) allow the manipulation of cell behavior for a limited period of time, e.g., triggering (trans)-differentiation by activation of lineage-specific molecular pathways. Some studies have already shown that alteration of gene expression using selected miRNAs can induce cardiac differentiation of MSC to a small extent [15,25,26], while data about mRNA-based cardiac reprogramming is still lacking.

Unlike multipotent MSCs, pluripotent stem cells (PSCs) have been demonstrated to efficiently differentiate into cardiomyocytes, characterized by a profound sarcomere organization and spontaneous beating behavior [27]. Yet, these PSC-derived cardiomyocytes typically still represent an immature cell type, resembling a neonatal cell stage rather than an adult phenotype [28,29]. The common cardiac programming approaches used to guide cardiac differentiation of PSCs mainly relies on the application of cytokines and small molecules [30,31]. However, PSCs bear tumorigenic risk due to genome modification (induced pluripotent stem cells, iPSC) and provoke ethical concerns (embryonic stem cells, ESC). Therefore, increasing the efficiency of cardiac programming of MSC would be beneficial for cardiovascular research, including their therapeutic use.

Here, we examined whether MSC derived from different sources, including bone marrow (BM), dental follicle and subcutaneous adipose tissue can be driven towards a cardiac lineage using a transient reprogramming strategy based on miRNA and mRNA transfection. According to our results, adipose tissue-derived MSC (adMSC) were found to be the most susceptible cell type for this reprogramming approach, as shown by enhanced expression of cardiac markers. At the same time, we observed the activation of transcriptome pathways involved in cardiac development following mRNA treatment.

#### **2. Material and Methods**

#### *2.1. Cell Culture*

BM-derived MSC (BM MSC) were obtained by sternal aspiration from donors undergone coronary bypass graft surgery. Anticoagulation was achieved by heparinization with 250 i.E./mL sodium heparin (Ratiopharm, Ulm, Germany). Mononuclear cells were isolated by density gradient centrifugation on 1077 Lymphocyte Separation Medium (LSM; PAA Laboratories, Pasching, Germany). MSC were enriched by plastic adherence and sub-cultured in MSC basal medium supplemented with SingleQuot (all Lonza, Cologne, Germany) and 1% Zellshield (Biochrom, Berlin, Germany).

Isolation of adMSC was performed by liposuction of healthy individuals. The extracted tissue was treated with collagenase for 30 min, followed by several filtrations and washing steps. The detailed process of adMSC isolation has been already described previously [32].

Dental follicle stem cells (DFSCs) were isolated from dental follicles of extracted wisdom teeth before tooth eruption. Following tooth removal, the follicle was removed and subjected to enzymatic treatment as presented earlier [33]. Upon tissue digestion, cells were seeded on tissue flasks and obtained by plastic adherence. DFSCs were maintained in DMEM-F12 (Thermo Fisher, Waltham, USA) supplemented with 10% FCS and 1% Zellshield.

All three types of stromal cells were maintained at 37 ◦C and 5% CO2 humidified atmosphere. Medium was changed every 2–3 days. Sub-cultivation was performed when cells reached a confluency of ~80–90%.

All donors have given their written consent for the donation of their tissue according to the Declaration of Helsinki. The study was approved by the ethical committee of the Medical Faculty of the University of Rostock (registration number: bone marrow A2010-23; renewal in 2015; adipose tissue: A2013-0112, renewal in 2019, dental tissue: A 2017-0158).

#### *2.2. Fluorescence-Activated Cell Sorting*

The expression of cell surface markers was quantified by flow cytometric analysis. Stromal cells were labelled with antibodies CD29-APC, CD44-PerCP-Cy5.5, CD45-V500, CD73-PE, CD117-PE-Cy7, PerCP-Cy5.5 CD90 (BD Biosciences, San Jose, USA), and CD105-AlexaFluor488 (AbD Serotec, Oxford, UK). Respective isotype antibodies served as negative controls. A measurement of 3 <sup>×</sup> <sup>10</sup><sup>4</sup> events was carried out using BD FACS LSRII flow cytometer (BD Biosciences).

To evaluate miRNA and mRNA uptake efficiency, cells were treated with different amounts of Cy3-labeled Pre-miRNA Negative Control #1 (AM17120, Thermo Fisher) or GFP-mRNA (Trilink, San Diego, USA) and analyzed by flow cytometry 24 h post transfection. To detect cytotoxicity, cells were labelled with Near-IR LIVE/DEAD fixable dead cell stain kit (Molecular Probes, Eugene, USA). Analysis of flow cytometry data, including gating, was conducted with the FACSDiva software, Version 8. (Becton Dickinson).

#### *2.3. Cardiac Reprogramming*

For cardiac reprogramming, 1 <sup>×</sup> 105 cells/well were seeded on 0.1% gelatin-coated 6 well plates and cultured to 80% confluency. We transfected 40 pmol of each miRNA (Pre-miR™ hsa-miR-1, Pre-miR™hsa-miR-499a-5p, Pre-miR™hsa-miR-208a-3p, Pre-miR™hsa-miR-133a-3p, all Thermo Fisher) with Lipofectamine® 2000 according to the manufacturer's instructions (Thermo Fisher). Transfection of custom-made mRNA (Trilink) was performed with Viromer Red® transfection reagent (Lipocalyx, Halle, Germany). Cells were either transfected with 2 μg MESP1 or with a combination of 1 μg GATA4, 1 μg MEF2C and 1 μg TBX5. One day after transfection of miRNA or mRNA, cells were subjected to two different medium conditions. For cardiac induction medium I (card ind. I), cells were incubated in RPMI, supplemented with B27 without insulin (Thermo fisher) for 7 days, followed by incubation in RPMI containing B27 +insulin/- vitamin A (Thermo Fisher) for another 21 days. Additionally, culture medium was supplemented with ascorbic acid (Sigma Aldrich, St. Louis, USA) and Wnt

pathway targeting small molecules, including 6 μM CHIR99021 (days 1–2), and 5 μM IWP-2 (days 4–5) (both Stemcell Technologies). For cardiac induction II (card ind II), a commercially available cardiomyocyte differentiation kit was used according to the instructions given by the manufacturer (Thermo Fisher, A2921201).

#### *2.4. IF Staining and Calcium Imaging*

To verify multipotency, BM-MSC, DFSC and adMSC were subjected to in vitro differentiation towards osteogenic, chondrogenic and adipogenic lineages using the Mesenchymal Stem Cell Functional Identification Kit (R & D). Differentiation was induced by maintaining cells under different culture conditions according to the manufacturer instructions for 20 days. Subsequently, cells were fluorescently labelled to detect fatty acid-binding protein 4 (FABP4), Aggrecan and Osteocalcin to visualize successful differentiation into adipocytes, chondrocytes, and osteocytes.

For labelling of cardiac markers, cells were seeded on coverslips and fixed with 4% PFA. Antibody staining was performed as described elsewhere [34]. Cells were labelled with anti sarcomeric α-actinin (abcam, ab9465), anti-NKX2.5 (Santa Cruz, sc-8697), anti-TBX5 (abcam, ab137833) and anti-MEF2C (Santa Cruz, sc-313).

To visualize intracellular calcium, cells were cultured on 8 well chamberslides (Ibidi). Three days after seeding, cells were incubated with the calcium sensitive dye Cal520 (AATBioquest) for one hour at 37 ◦C and subjected to fluorescence microscopy. All fluorescence images were acquired using Zeiss ELYRA LSM 780 (Zeiss, Oberkochen, Germany).
