Predicting mesenchymal stem cells potential for cardiac repair by clinical indicators and inducing differentiation to cardiomyocytes in vitro by mimicry of in vivo microenvironment

Predicting mesenchymal stem cells potential for cardiac repair by clinical indicators and inducing differentiation to cardiomyocytes in vitro by mimicry of in vivo microenvironment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Predicting mesenchymal stem cells potential for cardiac repair by clinical indicators and inducing differentiation to cardiomyocytes in vitro by mimicry of in vivo microenvironment Rose Alkhateeb, Serafima Romanova, Sandaara Kovalenko, Aleria Dolgodvorova, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7047558/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Patient-specific factors critically influence the therapeutic potential of bone marrow-derived mesenchymal stem cells (BM-MSCs) for cardiac regeneration. In this work, we identify lymphocyte count as a key clinical predictor of BM-MSC proliferative capacity, with coronary artery disease significantly delaying expansion in patients undergoing cardiac surgery. Thus, we established cell selection criteria for patient-derived MSCs for further differentiation into cardiomyocytes. To overcome the limitations of MSCs in cardiac differentiation, we developed a novel protocol using conditioned medium mimicking patient-specific in vivo conditions from iPSC-derived cardiomyocytes (iPSC-CM). This approach successfully generated functional cardiomyocytes from patient-specific BM-MSCs, as evidenced by spontaneous calcium transitions, structural maturation, and electrophysiological activity. Our results provide a unique protocol for inducing MSC differentiation for cell therapy from patient selection to patient-specific personalized preparation as a differentiation strategy. Health sciences/Cardiology Biological sciences/Cell biology Biological sciences/Stem cells Mesenchymal stem cells iPSC regenerative medicine cell therapy cardiac diseases patient data analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction The use of stem cell-based therapies has gained considerable interest due to the regenerative potential of stem cells. In particular, the therapeutic potential of mesenchymal stem cells (MSCs) in cardiac regeneration has become well established due to their multipotency, immunomodulatory properties, and relative availability from sources such as bone marrow (BM-MSCs) [ 1 – 7 ]. Clinical studies with MSC injection since the early 2000s have demonstrated their ability to improve cardiac function after infarction [ 8 – 10 ]. However, a critical obstacle to clinical application is the significant variability in therapeutic outcomes, often attributed to patient-specific factors affecting the efficiency of MSC proliferation and differentiation [ 11 – 13 ]. For example, donor age, comorbidities, and immune status may alter MSC functionality [ 12 , 14 , 16 ], but systematic analyses linking specific clinical parameters to MSC and BM-MSC behavior remain limited. Increasing evidence indicates that the systemic and local microenvironment critically regulates MSC fate, including proliferation and differentiation [ 15 ]. While clinical parameters (e.g., hemogram) reflect the patient's systemic state and potentially the bone marrow niche health, their direct predictive value for MSC expansion dynamics remains poorly characterized. Crucially, although immune interactions, particularly with lymphocytes, are known to modulate MSC function in vitro and in vivo [ 19 – 21 ], their impact on the fundamental proliferative capacity of patient-derived MSCs within a cardiac cohort is underexplored. Similarly, while studies suggest preserved angiogenic potential of MSCs in ischemic conditions [ 22 , 23 ], the direct effect of ischemic comorbidity (e.g., coronary artery disease) on MSC growth kinetics warrants systematic investigation. Therefore, establishing robust correlations between readily available clinical parameters (like lymphocyte count and CAD status) and the intrinsic proliferative potential of autologous BM-MSCs represents a critical step towards personalized therapy. In parallel, the successful use of MSCs for myocardial regeneration requires overcoming not only their patient specificity, but also the limited efficiency and directionality of their differentiation into cardiomyocytes in vitro and in vivo. Existing protocols (chemical inducers, genetic engineering) are often complex, insufficiently effective or unsafe [ 37 ]. Epigenetic reprogramming under the influence of microenvironmental factors is considered as a promising non-genetic approach to cell fate control [ 38 ]. Strategies to enhance MSC differentiation into functional cardiomyocytes, which are critical for structural repair, often rely on synthetic inducers or co-culture systems [ 30 – 35 ], which do not fully reproduce the in vivo microenvironment and patient-specific characteristics that affect the success of the differentiation itself and the survival of MSC cells even before differentiation. Thus, strategies that mimic the cardiac microenvironment using biological factors (e.g., conditioned media) appear to be a powerful tool for targeted differentiation of MSCs into functional cardiomyocytes, but are currently insufficiently reproducible and studied. In this study, we address two interrelated issues to improve the efficacy of autologous MSC therapy in CVD. First, we address the issue of identifying clinically relevant biomarkers to assess the proliferative (regenerative) potential of MSCs from CVD patients based on our collected patient database. We clearly demonstrate for the first time that a normal level of lymphocytes in a patient guarantees successful proliferation and growth of his MSCs in vitro, and therefore successful further differentiation. The second objective of the study was to develop an effective protocol for in vitro targeted differentiation of patient-derived BM-MSCs into functional cardiomyocytes by reprogramming their microenvironment using conditioned medium from iPSC-derived cardiomyocytes (iPSC-CM). For the first time, we not only mimic the in vivo environment in the efficient and patient-specific way, but also show that iPSC-CM conditioned medium effectively reprograms BM-MSCs, inducing their targeted differentiation into cells possessing key morphological, phenotypic and functional properties of mature cardiomyocytes (demonstrating spontaneous calcium oscillations and transverse striation), which opens the way to the creation of patient-specific cardiomyocytes for disease modeling and therapy screening. The obtained system of selection of MSCs based on clinical parameters and their initial induction into cardiomyocytes using the medium from patient-specific healthy IPSC-CM can be further used as a protocol for the preparation of cell therapy based on MSCs. 2. Results 2.1. Сharacterization of mesenchymal stem cells All obtained mesenchymal stem cells (MSCs) were characterized using immunohistochemical markers after isolation from bone marrow. A total of 18 lines were obtained, which were further analyzed in terms of clinical parameters depending on their proliferative properties. Immunofluorescence staining confirmed the expression of mesenchymal stem cell markers ALCAM (CD166) and Endoglin (CD140). After one week of culture on adhesion factor-coated glass, cells were fixed and stained. ALCAM (CD166) showed membrane localization with clear fluorescence. Endoglin (CD140) exhibited membrane-associated fluorescence. Nuclei were successfully stained with DAPI. Staining with Alexa Fluor 488 phalloidin successfully showed actin filaments (Fig. 1 ). Clinical and Biological Parameters Analysis Bone marrow samples were collected from 18 patients. BM-MSCs were isolated, cultured. The analysis of various clinical parameters and their relationship to BM-MSCs proliferation rate was performed. There is a clear relationship between lymphocyte count and the proliferation rate of cardiomyocytes (r = 0.779, p = 0.00037) (Fig. 2 A-B). Higher levels are linked to faster growth of MSCs leading to a quicker confluence. On the other hand, lower lymphocytes levels are associated with slower proliferation rate of MSCs with a marked increase in the time required to reach confluency. The data suggests that variation in MSC proliferation rate is more pronounced at higher lymphocytes levels. A moderate positive correlation between MSCs proliferation rate and coronary artery disease is shown (r = 0.59, p = 0.017) (Fig. 2 A-B). This suggests that the condition of ischemia is associated with slower proliferation rate. The scatter plot supports this finding, revealing that the samples of patients with ischemia have prolonged confluency time. Conversely, faster MSCs growth was seen in the case of the absence of ischemia. Non-significant correlation was observed between atherosclerosis and MSCs proliferation rate, (r = 0.31, p = 0.244) (Fig. 2 С). The same was found with hypertension (r = 0.333, p = 0.689), or age (r = 0.231, p = 0.389). These results suggest that these parameters do not impact the proliferation rate of MSCs. This study involved another patient’s MSC, not included in the overall statistics, where the patient had HIV (on therapy) and hepatitis C. He was excluded, since his cells did not proliferate. Thus, when selecting patients for MSC therapy, it is worth knowing in advance certain selected clinical parameters of the patient 2.2 MSC Differentiation After analyzing the data on MSC proliferation and the effect of patient parameters on the properties of MSCs, we modeled the process of MSC differentiation into cardiomyocytes after planting in the recipient's heart for cell therapy. Thus, we found a way to induce MSC differentiation into functional cardiomyocytes before transplantation, and also created a model for studying the behavior of MSCs when used as cell therapy. These two results can be used to improve the efficiency of MSC-based cell therapy. To induce MSC differentiation into cardiomyocytes, we used the so-called paracrine environment, maximally imitating the in vivo heart environment in a petri dish. For this, we used the medium obtained after obtaining cardiomyocytes during differentiation from iPSCs. We have previously demonstrated successful differentiation of human iPSC lines, including those used in this work [ 17 ]. These induced cardiomyocytes express factors corresponding to the environment we modeled, thereby inducing MSC differentiation. Such differentiation can check in advance whether the selected MSCs will differentiate into cardiomyocytes, and also give an impetus to their differentiation. Transplantation of MSCs already initiated for differentiation is possibly more effective in terms of the absence of arrhythmogenic effects The differentiation of MSCs into cardiomyocytes using the differentiation media collected from the parallel differentiation of IPSCs into cardiomyocytes was successfully achieved. Calcium imaging of MSCs-derived cardiomyocytes using Fluo4-AM showed a clear calcium transient which indicates the functionality of the cardiomyocytes. Moreover, it showed the fluorescent intensity across multiple cells (Fig. 3 A-B). We also analyzed the cells obtained during differentiation for cardiac markers, namely: myosin heavy chain, α-actinin, cardiac troponin. Some of the cells after differentiation demonstrated clear transverse striation of the cytoskeleton, characteristic of contractile cardiomyocytes (Fig. 3 C). Patch-clamp recording As part of this study, the electrophysiological activity of cardiomyocytes differentiated from MSCs was studied. The currents of the fast sodium channel INa, the L-type calcium channel ICa, L and the total current of the potassium channels IKv were recorded. After the differentiation of MSCs into cardiomyocytes, the general phenotype of the obtained cells was first checked. AP was recorded for these cardiomyocytes. The duration of AP at 80% repolarization (APD80) was 50 ± 4 ms (n = 4). An example of AP recording is shown in Fig. 4 A. During the process of differentiation of MSCs into cardiomyocytes, calcium, potassium and sodium currents were also recorded, they are presented in Fig. 4 B and Fig. 5 . INa and ICa,L were recorded by the patch-clamp method at room temperature using the step protocol depicted after differentiation (Fig. 4 B). Total potassium current IKv in cardiomyocytes obtained by differentiation from MSC. Figure 5 A shows the current IKv registered with I-V protocol (n = 4). By normalizing the amplitude of the currents on the cell capacity, after averaging, the amplitudes of all three currents were obtained in Fig. 5 . The average current density of INa was 7,66 ± 1,57 pA/pF (n = 3), ICa, L was 4,35 ± 1,92 pA/pF (n = 3) and IKv was 15,07 ± 6,06 pA/pF(n = 10). 3. Discussion The results of our study demonstrate a significant positive correlation between lymphocyte count and MSCs proliferation rate, suggesting the lymphocytes play a crucial role in modulating MSCs proliferation. This highlights the impact of the immune microenvironment on MSC behavior. These findings align with previous research results that indicated a close interaction between MSCs and the immune system. While this study focuses on the relationship between lymphocytes and MSCs proliferation, other studies have investigated different aspects of MSCs-immune system interactions. For instance, MSCs have been shown to modulate T cells and NK activity [ 19 , 20 ]. On the other hand, Spaggiari et al and Sotiropoulou et al [ 19 , 20 ] reported that cytokine-activated NK cells could lyse MSCs, while T- cells could induce MSCs apoptosis via Fas/FasL pathway [ 21 ]. The observed correlation between lymphocytes and MSCs proliferation highlights the importance of considering the immune-microenvironment in MSC-based therapies, as the presence of immune cells could influence the success of the therapy. Our data suggest that higher lymphocytes levels are associated with better MSCs growth, which potentially may lead to more efficient tissue engineering, where MSCs are often used. However the immune mediated lysis and apoptosis of MSCs, as reported by other studies, reverse this benefit. That is why more detailed analysis of the relationship between MSCs and lymphocytes is needed to enhance MSCs proliferation in the therapeutic clinical context. According to the results of our study, ischemic conditions negatively affect MSC growth and could impair MSC functionality. However, comparing our results with another study focusing on critical limb ischemia (CLI), some differences can be noted on how ischemia affects MSC function. Gremmels et al. reported that bone marrow–derived MSCs (BM-MSCs) from CLI patients show similar neovascularization capacity when compared to healthy controls [ 22 ]. These findings highlight the complexity of ischemic environments and their different effects on MSCs features. While ischemia may slow MSC proliferation, it doesn’t necessarily impair other aspects of MSC functionality like paracrine and proangiogenic effects [ 23 ]. Understanding these effects is important to optimize MSC-based therapy particularly for ischemic disease, where cell proliferation and angiogenesis play crucial roles in tissue recovery and healing. Our study showed no correlation between MSCs proliferation and atherosclerosis, which is consistent with the complexity of atherosclerosis pathophysiology. The current literature focuses on the therapeutic roles of MSCs in modulating inflammation, stabilizing plaques, and enhancing endothelial function. However, no studies have specifically examined the relationship between MSCs proliferation and atherosclerosis. This gap underlines the need for further investigation into the mechanisms by which MSCs, especially those derived from patients with atherosclerosis, exert their effects in atherosclerotic environments [ 24 ]. Despite no significant correlation between age and MSC proliferation rate, many studies indicate a reduction in MSC function and number with aging [ 25 ]. Researchers have reported that MSC niches from young individuals under 25 years old show better fibrillar organization, mechanical integrity, and responsiveness to growth factors, which contribute to higher proliferation rates [ 26 ]. In contrast, aging MSCs and their microenvironments often exhibit senescence-related changes and reduced proliferation capacity [ 27 ]. It is important to note that the absence of younger individuals, under 30 years old, may have impacted our results. Including younger participants in our study might have revealed a stronger correlation between MSC proliferation rate and age. Recently, stem cell transplantation to treat cardiac diseases has gained significant attention. Embryonic stem cells [ 28 ] and induced pluripotent stem cells (iPSCs) [ 29 ] have been successfully differentiated into cardiomyocytes. Additionally, bone marrow stem cells (BMSCs) are an easily accessible source with a high proliferative capacity, making them a promising option for cell therapy. Many methods for differentiating BMSCs into cardiomyocytes have been explored, including the use of chemicals [ 30 ], interleukins [ 31 ], and microRNAs[ 32 ]. Moreover, the microenvironment plays a crucial role in promoting differentiation. Studies have shown that culturing MSCs with specific substrates, like graphene[ 33 ] and collagen I nanomolecules [ 34 ], can induce specification into the cardiomyocyte lineage. Co-culturing BMSCs with cardiomyocytes has also been employed as a method for differentiation [ 35 ]. Another study used conditioned media from ischemic cardiac tissue to induce cardiac differentiation of MSCs[ 36 ]. In this study, conditioned media from cardiomyocyte-derived from IPSCs have been collected. This suggests that differentiation of MSCs into cardiomyocytes in the presence of the media conditioned by iPSCs-derived cardiomyocytes occurs through the exposure of MSCs to a specific cocktail that induces the differentiation towards the cardiac lineage. The importance of this is that the media collected during iPSC differentiation into cardiomyocytes mimics the in vivo environment that transplanted MSCs would encounter. The media contains a cocktail of signaling molecules, growth factors, and extracellular matrix components, which drive MSC differentiation toward the cardiac lineage. Moreover, the combined use of MSCs and iPSCs may have a promising role in cell therapy, as it provides both an optimal cell source and a supportive environment for cardiac regeneration. Our results also emphasize the importance of selecting MSCs that can proliferate efficiently, particularly in the context of differentiating them into cardiomyocytes. Understanding how biological factors affect MSC growth is crucial to optimize their use in cell therapy, leading to better therapeutic outcomes for cardiac disease patients. The obtained AP records showed that cardiomyocytes are not similar to ventricular ones. The resting potential of about − 50 mV and the short duration suggest that it is more like pacemaker cells (while no spontaneous activity was observed in the cells). According to the amplitude of the INa and ICaL currents, it can be said that these channels have not developed completely. Usually INa has amplitude for nA, however we registered hundreds pA. ICa,L is also less than the generally accepted data for healthy cells. Perhaps this is what causes the AP form. Total potassium currents IKv were lower than the standard ones, nevertheless, these currents were registered in almost every cell in which gigaomic contact was established (note that we did not see INa and ICa,L in all cells). It is suggested that the rest of the representatives of the family of potassium channels are also not fully developed during differentiation. However, additional further study of the channels is required. Our study has several limitations. The samples size may impact the strength of our findings, although the statistical variability appears sufficient to support our conclusions. However, a more extensive sample would improve robustness. Additionally, patient diversity within the study was limited, especially regarding age, potentially limiting the generalizability of our conclusions. The in vitro laboratory conditions may also not fully replicate in vivo environments, which can affect MSC proliferation and differentiation dynamics. This reinforces the importance of in vivo studies to confirm our findings. Our study has several limitations. The sample size may impact the strength of our results, although statistical variability appears to be sufficient to support our findings. However, a larger sample size would have increased robustness. Additionally, the diversity of patients in the study was limited, particularly with regard to age, potentially limiting the generalizability of our findings. In vitro laboratory conditions may also not fully replicate the in vivo environment, which may impact the dynamics of MSC proliferation and differentiation. This highlights the importance of further in vivo studies of our derived cells to validate our findings. Overall, this work provides new predictors of the regenerative potential of patient-derived bone marrow mesenchymal stem cells, as well as their ability to differentiate into cardiomyocytes. In doing so, we demonstrated the real functionality of differentiated cardiomyocytes from MSCs in vitro. In the future, this study will lead to qualitative patient selection using clinical markers and in vivo differentiation mimicry for more effective mesenchymal stem cell-based therapies for cardiac tissue injury. 4. Materials and Methods 4.1. Obtaining a bone marrow biopsy Samples were taken from 18 patients at the Moscow Regional Scientific Research Clinical Institute named after M.F. Vladimirsky. Clinical data were collected, including age, gender, body mass index (BMI), presence of coronary heart disease, atherosclerosis, and hypertension, as well as concentrations of glucose, lymphocytes, cholesterol, platelets, leukocytes, erythrocytes, neutrophils, and triglycerides. Bone marrow tissue samples were obtained from patients during open-heart surgery, immediately after sternotomy, by collecting bone marrow that had already been isolated from the sternum, in amounts of about 1–2 grams. The samples were placed in sterile test tubes and immediately processed to isolate BM-MSCs. 4.2. MSCs isolation Isolation of MSCs from bone tissue began by treating the biopsy with PBS, followed by cutting it into small pieces and incubating it with TrypLE Express (ThermoFisher Scientific) for 20 minutes at 37°C with 5% CO₂. The TrypLE Express was then inactivated by adding MSC expansion medium, Dulbecco's Modified Eagle Medium (DMEM) (ThermoFisher Scientific), followed by centrifugation at 1100g for 10 minutes. The resulting pellet was suspended in DMEM and cultured at a density of 1.5 × 10⁵ /cm². All flasks were coated with 0.1% gelatin (Biolot, Russia) for 30 minutes before seeding the MSCs. The MSC expansion medium was refreshed every three days. Confluence time was defined as the time required to reach over 80% confluency of adherent cells. Images of MSCs cultures were taken each three days using Olympus IX71 fluorescent microscope. 4.3. Immunocytochemistry A standard protocol for MSC immunostaining was used to study specific MSC markers, including ALCAM (CD166) and Endoglin (CD140). MSCs were dissociated using TrypLE Express, centrifuged at 200g for 7 minutes, and the resulting pellet was seeded on gelatin-coated glass dishes at a concentration of 50,000 cells per ml. After one week of culturing, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.4% Triton X-100 for 10 minutes. To stain F-actin, the cells were incubated with Alexa Fluor 488 goat anti-mouse IgG1 (A21121), 1:100 for one hour. The cells were then incubated with block buffer (1% bovine serum albumin in Phosphate Buffered Saline, PBS) for one hour at room temperature. Following this, the cells were incubated with primary antibodies overnight at 4°C, followed by secondary antibodies for 1 hour at room temperature. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Cells were washed twice with PBS after each incubation step. The cells were analyzed using an inverted fluorescence microscope (Olympus IX71, Japan). Primary antibodies and working dilutions: Endoglin (PAA980Hu02; CCC, USA;1 : 100); ALCAM (MAA002Hu22; CCC, USA; 1 :100); Secondary antibodies: (Sigma-Aldrich; working dilution, 1:400): Alexa Fluor 594 goat anti-rabbit IgG (H + L) CF™ (SAB4600107); Alexa Fluor 647 goat anti-rabbit IgG Atto (40839-1ML-F) 4.4. IPSCs differentiation and media collection In this study, we used m34sk3 cell line which was reprogrammed from a healthy donor and fully characterized previously [ 17 ]. Direct cardiac differentiation of m34sk3 was performed according to the original Gi-Wi protocol [ 18 ]. During changing the medium to a fresh one, the old media were collected to be used for MSCs differentiation. 4.5. MSCs differentiation MSCs were passaged and seeded onto a plate for differentiation into cardiomyocytes using media collected from induced pluripotent stem cell (iPSC)-derived cardiomyocytes. The differentiation process started by adding an equal volume of cardiomyocyte-conditioned media and RPMI 1640 medium (Lonza) containing B27 Supplement minus insulin (ThermoFisher Scientific),supplemented with fibroblast growth factor (FGF)(Sigma Aldrich) and non-essential amino acids (NEAA) (GIBCO,UK). On the following day, 5 µM IWP2 (Sigma Aldrich) was added to the culture. On day 3, the medium was replaced with a 1:1 mixture of RPMI 1640 containing B27 Supplement minus insulin and collected media from iPSC-derived cardiomyocytes, supplemented with 10 ng/mL bone morphogenetic protein 4 (BMP4)(Sigma Aldrich) and 10 ng/mL Activin A (Sigma Aldrich). Two days later, the medium was changed to collected media from iPSC-derived cardiomyocytes, with the addition of 5 µM IWP2. From day 10 onward, the medium was changed to RPMI 1640 containing B27 Supplement containing 10% fetal bovine serum (FBS) (Biosera, France). The protocol timeline is presented on Fig. 6 . 4.6. Calcium imaging Calcium imaging in cardiomyocytes-derived MSCs was done using calcium-dependent fluorescent dye Fluo4-AM. Cells were loaded with warmed RPMI 1640/B27 supplement containing 4 ug/ml Fluo4-AM at 37°C for 20 min. Then the medium was replaced with prewarmed Tyrodes’s solution solution (pH 7.4). Calcium dependent fluorescent was recorded for 30 s using an inverted fluorescent microscope (Olympus IX71) and its software and analyzed with the ImageJ program. 4.7. Patch-clamp assay The electrophysiological study was performed on cardiomyocytes differentiated from MSC. Currents INa, ICa.L and IKv were recorded at room temperature 24–25° C using the perforated “whole-cell” patch-clamp method. Amphotericin B (Sigma) at a concentration of 0.12 mg/mL was used as a perforating agent. The cardiac cells were placed in a chamber mounted on the slide of an Olympus IX71 inverted microscope. The chamber was perfused with an extracellular solution consisted of 150 mM NaCl, 1.8 mM CaCl2, 5.4 mM KCl, 1 mM MgCl2, 1 mM Na-pyruvate, 15 mM glucose, 15 mM HEPES (рН = 7.4 NaOH). Patch-clamp pipettes were filled with intracellular solution: 150 mM KCl, 5 mM NaCl, 2 mM CaCl2, 5 mM EGTA, 10 mM HEPES, 5 mM MgATP (рН = 7.2 KОН). The experiments were carried out using patch clamp installation consisting of the following main elements: digital converter Digidata 1440A (Axon Instruments, Inc., USA), amplifier Axopatch 200B (Axon Instruments, Inc., USA), MP-285 micromanipulator (Sutter Instrument), Olympus IX71 inverted microscope, Humbug noise filter (A-M-Systems), anti-vibration platform (AVTT75), temperature controller TC-324C (Warner Instruments). For the manufacture of pipettes: micropipette puller P-97 (Sutter Instrument), borosilicate glass (BF150-86-10, Sutter Instrument), microforge (MF-900, Narishige). Patch-clamp pipettes were made of borosilicate glass with a tip resistance of 2–4 MΩ when placed in the experimental solution. The pipette displacement was corrected to zero just before the formation of GΩ. After the gigaomic contact formation, a quick compensation adjustment of the amplifier instrument compensated for the pipette capacitance. Electrical access to the cell during perforation was marked by the appearance of slow capacitive currents that increased in amplitude as pores formed in the membrane with amphotericin. For recording INa and ICa,L stimulation protocol was used as prestep − 30 mV with duration of 150 ms and step 0 mV with duration of 300 ms. Total IKv current was recorded using step protocol from − 40 mV to + 60 mV with duration of 5 s. And for the I-V curve registration the protocol was from − 30 mV to + 60 mV with step for 15 mV for 2.5 s. AP was recorded using current-clamp configuration with 1 nA current stimulus with a duration of 2.5 ms. The membrane capacity measured using the Clamp 10.2 software ranged from 12 to 30 pF. Clampfit 10.2 (Molecular Devices) and Origin Pro 8.1 (Originlab Corporation) programs were used for data processing and analysis. The experimental data from the patch-clamp are presented in the form of a dependence of the amplitude of currents normalized by cell capacity on the potential - average values with a standard deviation. Averaging comes from at least three different cardiomyocytes. 4.10. Statistical and data analysis To investigate the relationship between monolayer confluency and the results of patient parameters, a correlation analysis was conducted. The Spearman correlation coefficient was calculated for continuous (quantitative) variables, including age, BMI, glucose, cholesterol, platelets, leukocytes, erythrocytes, neutrophils, and triglycerides. The biserial correlation formula was applied to binary variables, such as lymphocyte levels (normal/abnormal), sex, atherosclerosis, and coronary artery disease (CAD). For arterial hypertension, considered a discrete variable (stages of the disease), the correlation ratio was used. All calculations were performed using a code written in Python 3. Statistical analysis and visualization was performed by Seaborn(v0.13.2), Matplotlib(v3.7.1), SciPy(v1.13.1) and Statsmodels(v0.14.4). A p -value of less than 0.05 was considered significant for all analyses. 4.11. Ethical Approval All procedures were carried out in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and was conducted in accordance with the Declaration of Helsinki. The study was approved by the Ethics Committee of M.F. Vladimirsky Moscow Regional Clinical Research Institute (protocol №7 from 18.04.2024) and by the Moscow Institute of Physics and Technology Life Science Center Provisional Animal Care and Research Procedures Committee (Protocol No. A2-2012-09-02). 5. Conclusions Bone marrow-derived mesenchymal stem cells (BM-MSCs) have therapeutic potential for cardiac regeneration, but their efficacy depends on patient-specific factors. This study shows that lymphocyte counts strongly correlate with BM-MSC proliferation rate, while ischemic heart disease moderately delays growth in samples from 18 patients undergoing cardiac surgery. Importantly, we developed a biomimetic differentiation protocol using iPSC-derived cardiomyocyte medium (iPSC-CM-CM), which successfully generated functional cardiomyocytes from patient-derived BM-MSCs, mimicking in vivo conditions. Differentiated cells exhibited spontaneous calcium transients, striatal α-actinin expression, and key electrophysiological currents. These results identify lymphocyte levels as a clinical biomarker of MSC expansion potential and demonstrate iPSC-CM-CM as an effective tool for generating patient-specific cardiomyocytes, advancing personalized cardiac regeneration strategies. In essence, we have demonstrated for the first time the concept of selecting patients suitable for cell therapy of cardiac injury from selection to the onset of their differentiation into cardiomyocytes, demonstrating its feasibility. Declarations Author Contributions: Conceptualization, A. R., R. S., K.S., D.A., P.M., S.M., A.K.I., R.A.,Ts.V.A ; methodology, A. R., R. S., K.S., D.A., P.M., S.M., Ts.V.A; software, A. R., R. S., K.S., D.A., P.M., S.M., Ts.V.A; validation, A. R., S.M., Ts.V.A; formal analysis,; investigation, A. R., R. S., K.S., L.V., D.A., P.M., S.M., A.E., D.V., F.S., Z.D., Sh. D., A.K.I., Ts.V.A.; resources, A. R., D.A., S.M., Ts.V.A; data curation, A. R., D.A., S.M., Ts.V.A ; writing—original draft preparation, A. R., K.S., D.A., S.M., Ts.V.A ; writing—review and editing, L.V., A. R., R. S., K.S., D.A., P.M., S.M., A.K.I., Ts.V.A; visualization, A. R., K.S., D.A., S.M., Ts.V.A ; supervision, S.M., F.S., Z.D., Sh. D., A.K.I., Ts.V.A; project administration, S.M., A.K.I., R.A. Ts.V.A; funding acquisition, R.A., Ts.V., A.K.. All authors have read and agreed to the published version of the manuscript. Funding: The work was mainly supported by the Russian Science Foundation (RSF) (project No. 25-65-00037 of 22.05.2025). Institutional Review Board Statement: The study was approved by the Ethics Committee of M.F. Vladimirsky Moscow Regional Clinical Research Institute (protocol №7 from 18.04.2024) and by the Moscow Institute of Physics and Technology Life Science Center Provisional Animal Care and Research Procedures Committee (Protocol No. A2-2012-09-02). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: All raw data obtained or analyzed during this study are available in the Zenodo repository: https://doi.org/10.5281/zenodo.16446190. If additional data is required, you can obtain it by request by correspondence to the author: [email protected] Acknowledgments: The study was carried out within the framework of project No. 25-65-00037 of 22.05.2025 with the Russian Science Foundation (RSF). We would like to express special gratitude to the administration of the ITMO university, M.F. Vladimirsky Moscow Regional Clinical Research Institute and MIPT for the support of the authors. We express our gratitude to Berezhnoy A.K. for the assistance in visualizing the material, as well as to laboratory managers Dubrovskaya A.G. and Bakumenko S.S. Conflicts of Interest: The authors declare no conflicts of interest. References Ding, D. C. Shyu, and Shinn-Zong Lin. Mesenchymal stem cells. Cell Transplant. 20 (1), 5–14 (2011). Williams, A. R. & Joshua, M. Hare. 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Ischemic cardiac tissue conditioned media induced differentiation of human mesenchymal stem cells into early stage cardiomyocytes. Cytotechnology 64 , 563–575 (2012). Bui, T. et al. Challenges and limitations of strategies to promote therapeutic potential of human mesenchymal stem cells for cell-based cardiac repair. Korean circulation J. 51 (2), 97–113 (2021). Bhuvanalakshmi, G. et al. Epigenetic reprogramming converts human Wharton’s jelly mesenchymal stem cells into functional cardiomyocytes by differential regulation of Wnt mediators. Stem Cell Res. Ther. 8 , 1–15 (2017). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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02:08:26","extension":"xml","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":93715,"visible":true,"origin":"","legend":"","description":"","filename":"9d62d465f0ae4da19f44e0fbf35fa8f81structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7047558/v1/36abca246dfa8d7426af4a23.xml"},{"id":93537931,"identity":"3e892d04-8402-4330-a58d-a090c3cf2624","added_by":"auto","created_at":"2025-10-15 02:08:27","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":107227,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7047558/v1/3217b75fa77487a5e85d05e3.html"},{"id":93537912,"identity":"ba65face-f862-42ce-a5ac-7ce41a1ad89d","added_by":"auto","created_at":"2025-10-15 02:08:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":174796,"visible":true,"origin":"","legend":"\u003cp\u003eImmunofluorescence staining of BM-MSCs. (A) MSCs stained for ALCAM (CD166) in red. f-actin filaments are stained in green. DAPI is in blue. (B) MSCs stained for Endoglin (CD140) in red and for DAPI in blue. Scale bar: 100 µm.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7047558/v1/38970e2178a80b0633f4bdc5.png"},{"id":93537932,"identity":"5c01ebbe-7f90-4099-bd1e-ff63b450ff1a","added_by":"auto","created_at":"2025-10-15 02:08:28","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":84057,"visible":true,"origin":"","legend":"\u003cp\u003eClinical and biological factors affecting MSC proliferation. (A) Correlation heatmap of patient parameters (e.g., CAD, lymphocytes, age, BMI) with MSC confluency time. (B) Scatter plots showing strong positive correlation between lymphocyte count and MSC proliferation (r = 0.779), and moderate correlation with CAD status (r = 0.59); “NOT NORMAL” indicates abnormal clinical values. (C) Scatter plots showing weak correlations between MSC confluency time and atherosclerosis (r = 0.31), hypertension (r = 0.29). “NORMAL”/“NOT NORMAL” indicate disease status; blue dots represent patient samples.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7047558/v1/293739e0259f9e669f5aa30d.jpeg"},{"id":93537935,"identity":"c1cd3695-985c-435d-bb29-47db2f7c0c3b","added_by":"auto","created_at":"2025-10-15 02:08:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":414465,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional characterization of MSC-derived cardiomyocytes showing variable calcium transients via Fluo4-AM imaging: (A) linescan images; (B) corresponding traces; (C) α-actinin immunostaining (red) with Fast Fourier Transition (FFT) demonstrating the directionality of the transverse banding\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7047558/v1/fe95b3f4d246295d35b3177a.png"},{"id":93537910,"identity":"5b56acbe-0779-4dc5-920d-463ab6776f69","added_by":"auto","created_at":"2025-10-15 02:08:24","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":42020,"visible":true,"origin":"","legend":"\u003cp\u003eAction Potentials and Ionic Currents in MSC-Derived Cardiomyocytes. (A) Action potentials (APs) with APD80 of 50 ± 4 ms (n = 4) and patch-clamp recordings of INa and ICa,L using a step protocol at room temperature. (B) Representative traces of INa and ICa,L recorded in MSC-derived cardiomyocytes using a voltage step protocol, illustrating sodium and calcium channel activation.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7047558/v1/a334c357a66c4cc99a08897f.jpeg"},{"id":93537920,"identity":"64ccefca-da8b-4369-aea8-6ff4a72cde03","added_by":"auto","created_at":"2025-10-15 02:08:25","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":57190,"visible":true,"origin":"","legend":"\u003cp\u003eElectrophysiological Characterization of MSC-Derived Cardiomyocytes. (A) IKv recorded using an I–V step protocol. (B) I–V curve showing voltage-dependent activation of IKv (n = 4). (C) Average current densities.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7047558/v1/dfed8a8af1e85177f2be1b9b.jpeg"},{"id":93537904,"identity":"600529e8-81c5-4cac-a27f-3e9f97bbd8f8","added_by":"auto","created_at":"2025-10-15 02:08:23","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":67739,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the processes of differentiation of MSCs into cardiomyocytes using differentiation of iPSCs into cardiomyocytes\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7047558/v1/f5ae786767c9c95a4a3138df.jpeg"},{"id":97276256,"identity":"c77f0d5f-22d3-4f4e-b484-dd1832120962","added_by":"auto","created_at":"2025-12-02 15:53:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1615307,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7047558/v1/70bce200-fe40-401f-9156-a981de60c0a5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Predicting mesenchymal stem cells potential for cardiac repair by clinical indicators and inducing differentiation to cardiomyocytes in vitro by mimicry of in vivo microenvironment","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe use of stem cell-based therapies has gained considerable interest due to the regenerative potential of stem cells. In particular, the therapeutic potential of mesenchymal stem cells (MSCs) in cardiac regeneration has become well established due to their multipotency, immunomodulatory properties, and relative availability from sources such as bone marrow (BM-MSCs) [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Clinical studies with MSC injection since the early 2000s have demonstrated their ability to improve cardiac function after infarction [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, a critical obstacle to clinical application is the significant variability in therapeutic outcomes, often attributed to patient-specific factors affecting the efficiency of MSC proliferation and differentiation [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. For example, donor age, comorbidities, and immune status may alter MSC functionality [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], but systematic analyses linking specific clinical parameters to MSC and BM-MSC behavior remain limited.\u003c/p\u003e\u003cp\u003eIncreasing evidence indicates that the systemic and local microenvironment critically regulates MSC fate, including proliferation and differentiation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. While clinical parameters (e.g., hemogram) reflect the patient's systemic state and potentially the bone marrow niche health, their direct predictive value for MSC expansion dynamics remains poorly characterized. Crucially, although immune interactions, particularly with lymphocytes, are known to modulate MSC function in vitro and in vivo [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], their impact on the fundamental proliferative capacity of patient-derived MSCs within a cardiac cohort is underexplored. Similarly, while studies suggest preserved angiogenic potential of MSCs in ischemic conditions [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], the direct effect of ischemic comorbidity (e.g., coronary artery disease) on MSC growth kinetics warrants systematic investigation. Therefore, establishing robust correlations between readily available clinical parameters (like lymphocyte count and CAD status) and the intrinsic proliferative potential of autologous BM-MSCs represents a critical step towards personalized therapy.\u003c/p\u003e\u003cp\u003eIn parallel, the successful use of MSCs for myocardial regeneration requires overcoming not only their patient specificity, but also the limited efficiency and directionality of their differentiation into cardiomyocytes in vitro and in vivo. Existing protocols (chemical inducers, genetic engineering) are often complex, insufficiently effective or unsafe [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Epigenetic reprogramming under the influence of microenvironmental factors is considered as a promising non-genetic approach to cell fate control [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Strategies to enhance MSC differentiation into functional cardiomyocytes, which are critical for structural repair, often rely on synthetic inducers or co-culture systems [\u003cspan additionalcitationids=\"CR31 CR32 CR33 CR34\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], which do not fully reproduce the in vivo microenvironment and patient-specific characteristics that affect the success of the differentiation itself and the survival of MSC cells even before differentiation. Thus, strategies that mimic the cardiac microenvironment using biological factors (e.g., conditioned media) appear to be a powerful tool for targeted differentiation of MSCs into functional cardiomyocytes, but are currently insufficiently reproducible and studied.\u003c/p\u003e\u003cp\u003eIn this study, we address two interrelated issues to improve the efficacy of autologous MSC therapy in CVD. First, we address the issue of identifying clinically relevant biomarkers to assess the proliferative (regenerative) potential of MSCs from CVD patients based on our collected patient database. We clearly demonstrate for the first time that a normal level of lymphocytes in a patient guarantees successful proliferation and growth of his MSCs in vitro, and therefore successful further differentiation. The second objective of the study was to develop an effective protocol for in vitro targeted differentiation of patient-derived BM-MSCs into functional cardiomyocytes by reprogramming their microenvironment using conditioned medium from iPSC-derived cardiomyocytes (iPSC-CM). For the first time, we not only mimic the in vivo environment in the efficient and patient-specific way, but also show that iPSC-CM conditioned medium effectively reprograms BM-MSCs, inducing their targeted differentiation into cells possessing key morphological, phenotypic and functional properties of mature cardiomyocytes (demonstrating spontaneous calcium oscillations and transverse striation), which opens the way to the creation of patient-specific cardiomyocytes for disease modeling and therapy screening. The obtained system of selection of MSCs based on clinical parameters and their initial induction into cardiomyocytes using the medium from patient-specific healthy IPSC-CM can be further used as a protocol for the preparation of cell therapy based on MSCs.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Сharacterization of mesenchymal stem cells\u003c/h2\u003e\u003cp\u003eAll obtained mesenchymal stem cells (MSCs) were characterized using immunohistochemical markers after isolation from bone marrow. A total of 18 lines were obtained, which were further analyzed in terms of clinical parameters depending on their proliferative properties. Immunofluorescence staining confirmed the expression of mesenchymal stem cell markers ALCAM (CD166) and Endoglin (CD140). After one week of culture on adhesion factor-coated glass, cells were fixed and stained. ALCAM (CD166) showed membrane localization with clear fluorescence. Endoglin (CD140) exhibited membrane-associated fluorescence. Nuclei were successfully stained with DAPI. Staining with Alexa Fluor 488 phalloidin successfully showed actin filaments (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eClinical and Biological Parameters Analysis\u003c/em\u003e\u003c/p\u003e\u003cp\u003eBone marrow samples were collected from 18 patients. BM-MSCs were isolated, cultured. The analysis of various clinical parameters and their relationship to BM-MSCs proliferation rate was performed. There is a clear relationship between lymphocyte count and the proliferation rate of cardiomyocytes (r\u0026thinsp;=\u0026thinsp;0.779, p\u0026thinsp;=\u0026thinsp;0.00037) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). Higher levels are linked to faster growth of MSCs leading to a quicker confluence. On the other hand, lower lymphocytes levels are associated with slower proliferation rate of MSCs with a marked increase in the time required to reach confluency. The data suggests that variation in MSC proliferation rate is more pronounced at higher lymphocytes levels. A moderate positive correlation between MSCs proliferation rate and coronary artery disease is shown (r\u0026thinsp;=\u0026thinsp;0.59, p\u0026thinsp;=\u0026thinsp;0.017) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). This suggests that the condition of ischemia is associated with slower proliferation rate. The scatter plot supports this finding, revealing that the samples of patients with ischemia have prolonged confluency time. Conversely, faster MSCs growth was seen in the case of the absence of ischemia. Non-significant correlation was observed between atherosclerosis and MSCs proliferation rate, (r\u0026thinsp;=\u0026thinsp;0.31, p\u0026thinsp;=\u0026thinsp;0.244) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e С). The same was found with hypertension (r\u0026thinsp;=\u0026thinsp;0.333, p\u0026thinsp;=\u0026thinsp;0.689), or age (r\u0026thinsp;=\u0026thinsp;0.231, p\u0026thinsp;=\u0026thinsp;0.389). These results suggest that these parameters do not impact the proliferation rate of MSCs.\u003c/p\u003e\u003cp\u003eThis study involved another patient\u0026rsquo;s MSC, not included in the overall statistics, where the patient had HIV (on therapy) and hepatitis C. He was excluded, since his cells did not proliferate. Thus, when selecting patients for MSC therapy, it is worth knowing in advance certain selected clinical parameters of the patient\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003e2.2 MSC Differentiation\u003c/em\u003e\u003c/p\u003e\u003cp\u003eAfter analyzing the data on MSC proliferation and the effect of patient parameters on the properties of MSCs, we modeled the process of MSC differentiation into cardiomyocytes after planting in the recipient's heart for cell therapy. Thus, we found a way to induce MSC differentiation into functional cardiomyocytes before transplantation, and also created a model for studying the behavior of MSCs when used as cell therapy. These two results can be used to improve the efficiency of MSC-based cell therapy. To induce MSC differentiation into cardiomyocytes, we used the so-called paracrine environment, maximally imitating the in vivo heart environment in a petri dish. For this, we used the medium obtained after obtaining cardiomyocytes during differentiation from iPSCs. We have previously demonstrated successful differentiation of human iPSC lines, including those used in this work [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. These induced cardiomyocytes express factors corresponding to the environment we modeled, thereby inducing MSC differentiation. Such differentiation can check in advance whether the selected MSCs will differentiate into cardiomyocytes, and also give an impetus to their differentiation. Transplantation of MSCs already initiated for differentiation is possibly more effective in terms of the absence of arrhythmogenic effects\u003c/p\u003e\u003cp\u003eThe differentiation of MSCs into cardiomyocytes using the differentiation media collected from the parallel differentiation of IPSCs into cardiomyocytes was successfully achieved. Calcium imaging of MSCs-derived cardiomyocytes using Fluo4-AM showed a clear calcium transient which indicates the functionality of the cardiomyocytes. Moreover, it showed the fluorescent intensity across multiple cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). We also analyzed the cells obtained during differentiation for cardiac markers, namely: myosin heavy chain, α-actinin, cardiac troponin. Some of the cells after differentiation demonstrated clear transverse striation of the cytoskeleton, characteristic of contractile cardiomyocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003ePatch-clamp recording\u003c/em\u003e\u003c/p\u003e\u003cp\u003eAs part of this study, the electrophysiological activity of cardiomyocytes differentiated from MSCs was studied. The currents of the fast sodium channel INa, the L-type calcium channel ICa,\u003cem\u003eL\u003c/em\u003e and the total current of the potassium channels IKv were recorded.\u003c/p\u003e\u003cp\u003eAfter the differentiation of MSCs into cardiomyocytes, the general phenotype of the obtained cells was first checked. AP was recorded for these cardiomyocytes. The duration of AP at 80% repolarization (APD80) was 50\u0026thinsp;\u0026plusmn;\u0026thinsp;4 ms (n\u0026thinsp;=\u0026thinsp;4). An example of AP recording is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA. During the process of differentiation of MSCs into cardiomyocytes, calcium, potassium and sodium currents were also recorded, they are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. INa and ICa,L were recorded by the patch-clamp method at room temperature using the step protocol depicted after differentiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTotal potassium current IKv in cardiomyocytes obtained by differentiation from MSC. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA shows the current IKv registered with I-V protocol (n\u0026thinsp;=\u0026thinsp;4). By normalizing the amplitude of the currents on the cell capacity, after averaging, the amplitudes of all three currents were obtained in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The average current density of INa was 7,66\u0026thinsp;\u0026plusmn;\u0026thinsp;1,57 pA/pF (n\u0026thinsp;=\u0026thinsp;3), ICa, L was 4,35\u0026thinsp;\u0026plusmn;\u0026thinsp;1,92 pA/pF (n\u0026thinsp;=\u0026thinsp;3) and IKv was 15,07\u0026thinsp;\u0026plusmn;\u0026thinsp;6,06 pA/pF(n\u0026thinsp;=\u0026thinsp;10).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eThe results of our study demonstrate a significant positive correlation between lymphocyte count and MSCs proliferation rate, suggesting the lymphocytes play a crucial role in modulating MSCs proliferation. This highlights the impact of the immune microenvironment on MSC behavior. These findings align with previous research results that indicated a close interaction between MSCs and the immune system. While this study focuses on the relationship between lymphocytes and MSCs proliferation, other studies have investigated different aspects of MSCs-immune system interactions. For instance, MSCs have been shown to modulate T cells and NK activity [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. On the other hand, Spaggiari et al and Sotiropoulou et al [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] reported that cytokine-activated NK cells could lyse MSCs, while T- cells could induce MSCs apoptosis via Fas/FasL pathway [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The observed correlation between lymphocytes and MSCs proliferation highlights the importance of considering the immune-microenvironment in MSC-based therapies, as the presence of immune cells could influence the success of the therapy. Our data suggest that higher lymphocytes levels are associated with better MSCs growth, which potentially may lead to more efficient tissue engineering, where MSCs are often used. However the immune mediated lysis and apoptosis of MSCs, as reported by other studies, reverse this benefit. That is why more detailed analysis of the relationship between MSCs and lymphocytes is needed to enhance MSCs proliferation in the therapeutic clinical context.\u003c/p\u003e\u003cp\u003eAccording to the results of our study, ischemic conditions negatively affect MSC growth and could impair MSC functionality. However, comparing our results with another study focusing on critical limb ischemia (CLI), some differences can be noted on how ischemia affects MSC function. Gremmels et al. reported that bone marrow\u0026ndash;derived MSCs (BM-MSCs) from CLI patients show similar neovascularization capacity when compared to healthy controls [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These findings highlight the complexity of ischemic environments and their different effects on MSCs features. While ischemia may slow MSC proliferation, it doesn\u0026rsquo;t necessarily impair other aspects of MSC functionality like paracrine and proangiogenic effects [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Understanding these effects is important to optimize MSC-based therapy particularly for ischemic disease, where cell proliferation and angiogenesis play crucial roles in tissue recovery and healing.\u003c/p\u003e\u003cp\u003eOur study showed no correlation between MSCs proliferation and atherosclerosis, which is consistent with the complexity of atherosclerosis pathophysiology. The current literature focuses on the therapeutic roles of MSCs in modulating inflammation, stabilizing plaques, and enhancing endothelial function. However, no studies have specifically examined the relationship between MSCs proliferation and atherosclerosis. This gap underlines the need for further investigation into the mechanisms by which MSCs, especially those derived from patients with atherosclerosis, exert their effects in atherosclerotic environments [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDespite no significant correlation between age and MSC proliferation rate, many studies indicate a reduction in MSC function and number with aging [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Researchers have reported that MSC niches from young individuals under 25 years old show better fibrillar organization, mechanical integrity, and responsiveness to growth factors, which contribute to higher proliferation rates [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In contrast, aging MSCs and their microenvironments often exhibit senescence-related changes and reduced proliferation capacity [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. It is important to note that the absence of younger individuals, under 30 years old, may have impacted our results. Including younger participants in our study might have revealed a stronger correlation between MSC proliferation rate and age.\u003c/p\u003e\u003cp\u003eRecently, stem cell transplantation to treat cardiac diseases has gained significant attention. Embryonic stem cells [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and induced pluripotent stem cells (iPSCs) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] have been successfully differentiated into cardiomyocytes. Additionally, bone marrow stem cells (BMSCs) are an easily accessible source with a high proliferative capacity, making them a promising option for cell therapy. Many methods for differentiating BMSCs into cardiomyocytes have been explored, including the use of chemicals [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], interleukins [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and microRNAs[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Moreover, the microenvironment plays a crucial role in promoting differentiation. Studies have shown that culturing MSCs with specific substrates, like graphene[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and collagen I nanomolecules [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], can induce specification into the cardiomyocyte lineage. Co-culturing BMSCs with cardiomyocytes has also been employed as a method for differentiation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Another study used conditioned media from ischemic cardiac tissue to induce cardiac differentiation of MSCs[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In this study, conditioned media from cardiomyocyte-derived from IPSCs have been collected. This suggests that differentiation of MSCs into cardiomyocytes in the presence of the media conditioned by iPSCs-derived cardiomyocytes occurs through the exposure of MSCs to a specific cocktail that induces the differentiation towards the cardiac lineage. The importance of this is that the media collected during iPSC differentiation into cardiomyocytes mimics the in vivo environment that transplanted MSCs would encounter. The media contains a cocktail of signaling molecules, growth factors, and extracellular matrix components, which drive MSC differentiation toward the cardiac lineage. Moreover, the combined use of MSCs and iPSCs may have a promising role in cell therapy, as it provides both an optimal cell source and a supportive environment for cardiac regeneration. Our results also emphasize the importance of selecting MSCs that can proliferate efficiently, particularly in the context of differentiating them into cardiomyocytes. Understanding how biological factors affect MSC growth is crucial to optimize their use in cell therapy, leading to better therapeutic outcomes for cardiac disease patients.\u003c/p\u003e\u003cp\u003eThe obtained AP records showed that cardiomyocytes are not similar to ventricular ones. The resting potential of about \u0026minus;\u0026thinsp;50 mV and the short duration suggest that it is more like pacemaker cells (while no spontaneous activity was observed in the cells). According to the amplitude of the INa and ICaL currents, it can be said that these channels have not developed completely. Usually INa has amplitude for nA, however we registered hundreds pA. ICa,L is also less than the generally accepted data for healthy cells. Perhaps this is what causes the AP form. Total potassium currents IKv were lower than the standard ones, nevertheless, these currents were registered in almost every cell in which gigaomic contact was established (note that we did not see INa and ICa,L in all cells). It is suggested that the rest of the representatives of the family of potassium channels are also not fully developed during differentiation. However, additional further study of the channels is required.\u003c/p\u003e\u003cp\u003eOur study has several limitations. The samples size may impact the strength of our findings, although the statistical variability appears sufficient to support our conclusions. However, a more extensive sample would improve robustness. Additionally, patient diversity within the study was limited, especially regarding age, potentially limiting the generalizability of our conclusions. The in vitro laboratory conditions may also not fully replicate in vivo environments, which can affect MSC proliferation and differentiation dynamics. This reinforces the importance of in vivo studies to confirm our findings.\u003c/p\u003e\u003cp\u003eOur study has several limitations. The sample size may impact the strength of our results, although statistical variability appears to be sufficient to support our findings. However, a larger sample size would have increased robustness. Additionally, the diversity of patients in the study was limited, particularly with regard to age, potentially limiting the generalizability of our findings. In vitro laboratory conditions may also not fully replicate the in vivo environment, which may impact the dynamics of MSC proliferation and differentiation. This highlights the importance of further in vivo studies of our derived cells to validate our findings.\u003c/p\u003e\u003cp\u003eOverall, this work provides new predictors of the regenerative potential of patient-derived bone marrow mesenchymal stem cells, as well as their ability to differentiate into cardiomyocytes. In doing so, we demonstrated the real functionality of differentiated cardiomyocytes from MSCs in vitro. In the future, this study will lead to qualitative patient selection using clinical markers and in vivo differentiation mimicry for more effective mesenchymal stem cell-based therapies for cardiac tissue injury.\u003c/p\u003e"},{"header":"4. Materials and Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e4.1. Obtaining a bone marrow biopsy\u003c/h2\u003e\u003cp\u003eSamples were taken from 18 patients at the Moscow Regional Scientific Research Clinical Institute named after M.F. Vladimirsky. Clinical data were collected, including age, gender, body mass index (BMI), presence of coronary heart disease, atherosclerosis, and hypertension, as well as concentrations of glucose, lymphocytes, cholesterol, platelets, leukocytes, erythrocytes, neutrophils, and triglycerides. Bone marrow tissue samples were obtained from patients during open-heart surgery, immediately after sternotomy, by collecting bone marrow that had already been isolated from the sternum, in amounts of about 1\u0026ndash;2 grams. The samples were placed in sterile test tubes and immediately processed to isolate BM-MSCs.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e4.2. MSCs isolation\u003c/h2\u003e\u003cp\u003eIsolation of MSCs from bone tissue began by treating the biopsy with PBS, followed by cutting it into small pieces and incubating it with TrypLE Express (ThermoFisher Scientific) for 20 minutes at 37\u0026deg;C with 5% CO₂. The TrypLE Express was then inactivated by adding MSC expansion medium, Dulbecco's Modified Eagle Medium (DMEM) (ThermoFisher Scientific), followed by centrifugation at 1100g for 10 minutes. The resulting pellet was suspended in DMEM and cultured at a density of 1.5 \u0026times; 10⁵ /cm\u0026sup2;. All flasks were coated with 0.1% gelatin (Biolot, Russia) for 30 minutes before seeding the MSCs. The MSC expansion medium was refreshed every three days. Confluence time was defined as the time required to reach over 80% confluency of adherent cells. Images of MSCs cultures were taken each three days using Olympus IX71 fluorescent microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e4.3. Immunocytochemistry\u003c/h2\u003e\u003cp\u003eA standard protocol for MSC immunostaining was used to study specific MSC markers, including ALCAM (CD166) and Endoglin (CD140). MSCs were dissociated using TrypLE Express, centrifuged at 200g for 7 minutes, and the resulting pellet was seeded on gelatin-coated glass dishes at a concentration of 50,000 cells per ml. After one week of culturing, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.4% Triton X-100 for 10 minutes. To stain F-actin, the cells were incubated with Alexa Fluor 488 goat anti-mouse IgG1 (A21121), 1:100 for one hour. The cells were then incubated with block buffer (1% bovine serum albumin in Phosphate Buffered Saline, PBS) for one hour at room temperature. Following this, the cells were incubated with primary antibodies overnight at 4\u0026deg;C, followed by secondary antibodies for 1 hour at room temperature. Nuclei were stained with 4\u0026prime;,6-diamidino-2-phenylindole (DAPI). Cells were washed twice with PBS after each incubation step. The cells were analyzed using an inverted fluorescence microscope (Olympus IX71, Japan).\u003c/p\u003e\u003cp\u003ePrimary antibodies and working dilutions: Endoglin (PAA980Hu02; CCC, USA;1 : 100); ALCAM (MAA002Hu22; CCC, USA; 1 :100); Secondary antibodies: (Sigma-Aldrich; working dilution, 1:400): Alexa Fluor 594 goat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) CF\u0026trade; (SAB4600107); Alexa Fluor 647 goat anti-rabbit IgG Atto (40839-1ML-F)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e4.4. IPSCs differentiation and media collection\u003c/h2\u003e\u003cp\u003eIn this study, we used m34sk3 cell line which was reprogrammed from a healthy donor and fully characterized previously [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Direct cardiac differentiation of m34sk3 was performed according to the original Gi-Wi protocol [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. During changing the medium to a fresh one, the old media were collected to be used for MSCs differentiation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e4.5. MSCs differentiation\u003c/h2\u003e\u003cp\u003eMSCs were passaged and seeded onto a plate for differentiation into cardiomyocytes using media collected from induced pluripotent stem cell (iPSC)-derived cardiomyocytes. The differentiation process started by adding an equal volume of cardiomyocyte-conditioned media and RPMI 1640 medium (Lonza) containing B27 Supplement minus insulin (ThermoFisher Scientific),supplemented with fibroblast growth factor (FGF)(Sigma Aldrich) and non-essential amino acids (NEAA) (GIBCO,UK). On the following day, 5 \u0026micro;M IWP2 (Sigma Aldrich) was added to the culture. On day 3, the medium was replaced with a 1:1 mixture of RPMI 1640 containing B27 Supplement minus insulin and collected media from iPSC-derived cardiomyocytes, supplemented with 10 ng/mL bone morphogenetic protein 4 (BMP4)(Sigma Aldrich) and 10 ng/mL Activin A (Sigma Aldrich). Two days later, the medium was changed to collected media from iPSC-derived cardiomyocytes, with the addition of 5 \u0026micro;M IWP2. From day 10 onward, the medium was changed to RPMI 1640 containing B27 Supplement containing 10% fetal bovine serum (FBS) (Biosera, France). The protocol timeline is presented on Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e4.6. Calcium imaging\u003c/h2\u003e\u003cp\u003eCalcium imaging in cardiomyocytes-derived MSCs was done using calcium-dependent fluorescent dye Fluo4-AM. Cells were loaded with warmed RPMI 1640/B27 supplement containing 4 ug/ml Fluo4-AM at 37\u0026deg;C for 20 min. Then the medium was replaced with prewarmed Tyrodes\u0026rsquo;s solution solution (pH 7.4). Calcium dependent fluorescent was recorded for 30 s using an inverted fluorescent microscope (Olympus IX71) and its software and analyzed with the ImageJ program.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e4.7. Patch-clamp assay\u003c/h2\u003e\u003cp\u003eThe electrophysiological study was performed on cardiomyocytes differentiated from MSC. Currents INa, ICa.L and IKv were recorded at room temperature 24\u0026ndash;25\u0026deg; C using the perforated \u0026ldquo;whole-cell\u0026rdquo; patch-clamp method. Amphotericin B (Sigma) at a concentration of 0.12 mg/mL was used as a perforating agent. The cardiac cells were placed in a chamber mounted on the slide of an Olympus IX71 inverted microscope. The chamber was perfused with an extracellular solution consisted of 150 mM NaCl, 1.8 mM CaCl2, 5.4 mM KCl, 1 mM MgCl2, 1 mM Na-pyruvate, 15 mM glucose, 15 mM HEPES (рН = 7.4 NaOH). Patch-clamp pipettes were filled with intracellular solution: 150 mM KCl, 5 mM NaCl, 2 mM CaCl2, 5 mM EGTA, 10 mM HEPES, 5 mM MgATP (рН = 7.2 KОН).\u003c/p\u003e\u003cp\u003eThe experiments were carried out using patch clamp installation consisting of the following main elements: digital converter Digidata 1440A (Axon Instruments, Inc., USA), amplifier Axopatch 200B (Axon Instruments, Inc., USA), MP-285 micromanipulator (Sutter Instrument), Olympus IX71 inverted microscope, Humbug noise filter (A-M-Systems), anti-vibration platform (AVTT75), temperature controller TC-324C (Warner Instruments). For the manufacture of pipettes: micropipette puller P-97 (Sutter Instrument), borosilicate glass (BF150-86-10, Sutter Instrument), microforge (MF-900, Narishige). Patch-clamp pipettes were made of borosilicate glass with a tip resistance of 2\u0026ndash;4 MΩ when placed in the experimental solution. The pipette displacement was corrected to zero just before the formation of GΩ. After the gigaomic contact formation, a quick compensation adjustment of the amplifier instrument compensated for the pipette capacitance. Electrical access to the cell during perforation was marked by the appearance of slow capacitive currents that increased in amplitude as pores formed in the membrane with amphotericin.\u003c/p\u003e\u003cp\u003eFor recording INa and ICa,L stimulation protocol was used as prestep \u0026minus;\u0026thinsp;30 mV with duration of 150 ms and step 0 mV with duration of 300 ms. Total IKv current was recorded using step protocol from \u0026minus;\u0026thinsp;40 mV to +\u0026thinsp;60 mV with duration of 5 s. And for the I-V curve registration the protocol was from \u0026minus;\u0026thinsp;30 mV to +\u0026thinsp;60 mV with step for 15 mV for 2.5 s. AP was recorded using current-clamp configuration with 1 nA current stimulus with a duration of 2.5 ms.\u003c/p\u003e\u003cp\u003eThe membrane capacity measured using the Clamp 10.2 software ranged from 12 to 30 pF. Clampfit 10.2 (Molecular Devices) and Origin Pro 8.1 (Originlab Corporation) programs were used for data processing and analysis. The experimental data from the patch-clamp are presented in the form of a dependence of the amplitude of currents normalized by cell capacity on the potential - average values with a standard deviation. Averaging comes from at least three different cardiomyocytes.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e4.10. Statistical and data analysis\u003c/h2\u003e\u003cp\u003eTo investigate the relationship between monolayer confluency and the results of patient parameters, a correlation analysis was conducted. The Spearman correlation coefficient was calculated for continuous (quantitative) variables, including age, BMI, glucose, cholesterol, platelets, leukocytes, erythrocytes, neutrophils, and triglycerides. The biserial correlation formula was applied to binary variables, such as lymphocyte levels (normal/abnormal), sex, atherosclerosis, and coronary artery disease (CAD). For arterial hypertension, considered a discrete variable (stages of the disease), the correlation ratio was used. All calculations were performed using a code written in Python 3. Statistical analysis and visualization was performed by Seaborn(v0.13.2), Matplotlib(v3.7.1), SciPy(v1.13.1) and Statsmodels(v0.14.4). A \u003cem\u003ep\u003c/em\u003e-value of less than 0.05 was considered significant for all analyses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e4.11. Ethical Approval\u003c/h2\u003e\u003cp\u003e All procedures were carried out in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and was conducted in accordance with the Declaration of Helsinki. The study was approved by the Ethics Committee of M.F. Vladimirsky Moscow Regional Clinical Research Institute (protocol №7 from 18.04.2024) and by the Moscow Institute of Physics and Technology Life Science Center Provisional Animal Care and Research Procedures Committee (Protocol No. A2-2012-09-02).\u003c/p\u003e\u003c/div\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eBone marrow-derived mesenchymal stem cells (BM-MSCs) have therapeutic potential for cardiac regeneration, but their efficacy depends on patient-specific factors. This study shows that lymphocyte counts strongly correlate with BM-MSC proliferation rate, while ischemic heart disease moderately delays growth in samples from 18 patients undergoing cardiac surgery. Importantly, we developed a biomimetic differentiation protocol using iPSC-derived cardiomyocyte medium (iPSC-CM-CM), which successfully generated functional cardiomyocytes from patient-derived BM-MSCs, mimicking in vivo conditions. Differentiated cells exhibited spontaneous calcium transients, striatal α-actinin expression, and key electrophysiological currents. These results identify lymphocyte levels as a clinical biomarker of MSC expansion potential and demonstrate iPSC-CM-CM as an effective tool for generating patient-specific cardiomyocytes, advancing personalized cardiac regeneration strategies. In essence, we have demonstrated for the first time the concept of selecting patients suitable for cell therapy of cardiac injury from selection to the onset of their differentiation into cardiomyocytes, demonstrating its feasibility.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Conceptualization, A. R., R. S., K.S., D.A., P.M., S.M., A.K.I., R.A.,Ts.V.A ; methodology, A. R., R. S., K.S., D.A., P.M., S.M., Ts.V.A; software, A. R., R. S., K.S., D.A., P.M., S.M., Ts.V.A; validation, A. R., S.M., Ts.V.A; formal analysis,; investigation, A. R., R. S., K.S., L.V., D.A., P.M., S.M., A.E., D.V., F.S., Z.D., Sh. D., A.K.I., Ts.V.A.; resources, A. R., D.A., S.M., Ts.V.A; data curation, A. R., D.A., S.M., Ts.V.A ; writing\u0026mdash;original draft preparation, A. R., K.S., D.A., S.M., Ts.V.A ; writing\u0026mdash;review and editing, L.V., A. R., R. S., K.S., D.A., P.M., S.M., A.K.I., Ts.V.A; visualization, A. R., K.S., D.A., S.M., Ts.V.A ; supervision, S.M., F.S., Z.D., Sh. D., A.K.I., Ts.V.A; project administration, S.M., A.K.I., R.A. Ts.V.A; funding acquisition, R.A., Ts.V., A.K.. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The work was mainly supported by the Russian Science Foundation (RSF) (project No. 25-65-00037 of 22.05.2025).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u003c/strong\u003e The study was approved by the Ethics Committee of M.F. Vladimirsky Moscow Regional Clinical Research Institute (protocol №7 from 18.04.2024) and by the Moscow Institute of Physics and Technology Life Science Center Provisional Animal Care and Research Procedures Committee (Protocol No. A2-2012-09-02).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u0026nbsp;\u003c/strong\u003eInformed consent was obtained from all subjects involved in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll raw data obtained or analyzed during this study are available in the Zenodo repository: https://doi.org/10.5281/zenodo.16446190. If additional data is required, you can obtain it by request by correspondence to the author: [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eThe study was carried out within the framework of project No. 25-65-00037 of 22.05.2025 with the Russian Science Foundation (RSF). We would like to express special gratitude to the administration of the ITMO university, M.F. Vladimirsky Moscow Regional Clinical Research Institute \u0026nbsp;and MIPT for the support of the authors. We express our gratitude to Berezhnoy A.K. for the assistance in visualizing the material, as well as to laboratory managers Dubrovskaya A.G. and Bakumenko S.S.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDing, D. C. Shyu, and Shinn-Zong Lin. 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Ther.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 1\u0026ndash;15 (2017).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Mesenchymal stem cells, iPSC, regenerative medicine, cell therapy, cardiac diseases, patient data analysis","lastPublishedDoi":"10.21203/rs.3.rs-7047558/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7047558/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePatient-specific factors critically influence the therapeutic potential of bone marrow-derived mesenchymal stem cells (BM-MSCs) for cardiac regeneration. In this work, we identify lymphocyte count as a key clinical predictor of BM-MSC proliferative capacity, with coronary artery disease significantly delaying expansion in patients undergoing cardiac surgery. Thus, we established cell selection criteria for patient-derived MSCs for further differentiation into cardiomyocytes. To overcome the limitations of MSCs in cardiac differentiation, we developed a novel protocol using conditioned medium mimicking patient-specific in vivo conditions from iPSC-derived cardiomyocytes (iPSC-CM). This approach successfully generated functional cardiomyocytes from patient-specific BM-MSCs, as evidenced by spontaneous calcium transitions, structural maturation, and electrophysiological activity. Our results provide a unique protocol for inducing MSC differentiation for cell therapy from patient selection to patient-specific personalized preparation as a differentiation strategy.\u003c/p\u003e","manuscriptTitle":"Predicting mesenchymal stem cells potential for cardiac repair by clinical indicators and inducing differentiation to cardiomyocytes in vitro by mimicry of in vivo microenvironment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-15 02:08:09","doi":"10.21203/rs.3.rs-7047558/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"597c5a77-41ec-41e8-bf06-be954fe642ed","owner":[],"postedDate":"October 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":56043698,"name":"Health sciences/Cardiology"},{"id":56043699,"name":"Biological sciences/Cell biology"},{"id":56043700,"name":"Biological sciences/Stem cells"}],"tags":[],"updatedAt":"2025-12-02T15:53:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-15 02:08:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7047558","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7047558","identity":"rs-7047558","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}