Effect of the dose, timing, and route of administration of mesenchymal stem cells on their regenerative capacity after myocardial infarction: A systematic review

Introduction

The global rise in acute myocardial infarction (AMI) cases poses a significant health challenge. In China, it is estimated that by 2030 there will be 23 million cases,¹ with high mortality rates persisting despite technological advances in its treatment. In Japan, AMI with ST segment elevation has a mortality rate up to 12.3%.² Although survival can reach up to 85%, one-year post-AMI,³ the consequences can severely impact quality of life, underscoring the need to explore alternative treatments to mitigate these effects.

Adult stem cells have gained great importance in recent years due to their potential to promote the regeneration of cardiac tissue. Among them, multipotent mesenchymal stromal cells (MSCs) stand out, which were described in 1967 by Friedenstein et al, who isolated from the bone marrow (BM), adherent clonogenic cells like fibroblasts called fibroblast colony-forming units (CFU-F). The International Society for Cellular Therapy (ISCT) established criteria to identify them, in 2006; First, MSCs must adhere to plastic when maintained under standard culture conditions, second, MSCs must express CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79-α or CD19, and HLA-DR surface molecules. Third, MSCs must differentiate into osteoblasts, adipocytes, and chondroblasts in vitro.⁴

There are multiple sources of obtaining MSCs. Those derived from bone marrow (BM-MSCs) are some of the most studied, and can be expanded in vitro.¹ Furthermore, when they have been applied in vivo, they have differentiated into cardiomyocytes⁵ and produce cytokines, chemokines, and micro RNAs (miRNAs) to reduce inflammation and remodeling of the infarcted area.¹

Another source currently used to obtain MSCs is adipose tissue, which can also be easily expanded in vitro. These cells (ADSCs) present properties similar to BM-MSCs and have demonstrated their ability to reduce cardiac remodeling and promote angiogenesis. These effects have been demonstrated in a porcine model of AMI in which less fibrosis was demonstrated in the infarcted area, evaluated with Picrosirius red, as well as greater vascularization in the peri-infarct area, detected by anti-CD31 antibodies labeled with fluorescent substances.⁶

Zhang et al., 2021, conducted a single-blind multicenter randomized controlled trial, that evaluated 43 patients with AMI treated with coronary reperfusion (PCI), 21 of them received 3.31 ± 1.70 × 10⁶ BM-MSCs, between 23.24 ± 7.69 days, after the AMI, and 22 patients, usual post-PCI care. No significant differences were found in left ventricular end-systolic volume (LVESV), end-diastolic volume (LVEDV), and left ventricular ejection fraction (LVEF) at six months of follow-up, between both groups. Nor did it detect differences in myocardial perfusion and the rate of metabolic defects at twelve months. They concluded that the sample number was small, the dose applied was insufficient, the application time was late, and that the route of administration and the source of obtaining of the MSCs were probably not optimal.¹

Gathier, W.A. et al., 2019, conducted an experimental study to compare the retention percentage of MSCs radiolabeled with Indium-111, applied by retrograde coronary venous infusion (RCVI) or by PCI in a porcine model of AMI; Likewise, the safety of the procedures was evaluated. Four weeks after AMI was induced, MSCs were applied to six pigs in each group [median age and weight at the time of AMI, 20 weeks (IQR: 18-22) and 72 kilograms (IQR: 68-76), respectively and four hours later, in vivo and ex vivo scintigraphy was performed, which made it possible to evaluate the percentage of retention of the MSCs by dividing the radioactivity of the heart by the total radioactivity emitted by the body or by the sum of those emitted by the independent organs, respectively. The median number of viable MSCs administered was 2.4 ×10⁶ (IQR: 1.6–2.4) in the PCI group versus 1.6 × 10⁶ (IQR: 1.3–1.7) in the RCVI group (p=0.167). in vivo retention presented a significant difference between both groups with a median of 2.89% (IQR: 2.14–3.86) in the RCVI group versus 13.74% (IQR: 10.20–15.41) in the PCI group (p=0.002). Median ex vivo retention was 2.55% (IQR: 1.86-3.16) in the RCVI group versus 39.40% (IQR: 38.54-44.64) in the PCI group (p=0.002).

In the RCVI group, coronary sinus (CS) dissection was observed in three of the six pigs at the level of the tip of the catheter, three animals presented a small to moderate clear pericardial effusion and a hematoma of approximately 4 cm² in the atrioventricular groove on the left side. One animal in the PCI group showed no flow directly after infusion of the cells, probably due to thrombus formation. Flow was restored 5 min after angioplasty.⁷

Mori D et al., 2018, evaluated the regenerative effects of human ADSCs using a porcine model of AMI, treated 6 pigs with 10⁸ ADSCs applied to the epicardium, using a spray containing a fibrinogen matrix, and another 6 served as controls. At four months, LVEF was higher in the experimental group (p < 0.05),⁶ and left ventricular end-systolic diameter (LVDs) was lower in the same group. The end-systolic and end-diastolic volume/pressure ratio, as well as the global coronary flow reserve, was better preserved in the experimental group, after the same period.

At 8 weeks after transplantation, less fibrosis in the infarcted area, a lower number of hypertrophic cardiomyocytes, and greater vascularization in the peri-infarct region were observed in the group treated with ADSCs evaluated with Masson’s trichrome staining, PAS staining, and immunohistochemistry for anti-CD31, respectively. Finally, a higher concentration of basic fibroblast growth factor (b FGF), vascular endothelial growth factor (VEGF) and stromal cell-derived factor 1 (SDF-1) was detected. The authors concluded that spray application of ADSCs is safe and promotes the recovery of cardiac function.⁶

Su Hyun K et al., 2018, conducted a randomized, single-blind trial, evaluating 26 patients with AMI with ST segment elevation, treated by PCI, 14 of whom were randomized to the experimental group treated with BM MSCs and 12 patients to the control group. The experimental group received 7.2 ± 0.9 × 10⁷ BM - MSCs, 30 days after PCI, and the control group received standardized care. Patients were evaluated 4 and 12 months after PCI, using single photon emission tomography (SPECT) and echocardiography, respectively; detecting significant differences in LVEF between both groups at 4 months (8.8 ± 2.9 vs. 4.8 ± 1.9%, p = 0.031) and 12 months (9.9% ± 5.2 vs. 6.5% ± 2.7%, p = 0.048). Likewise, the cells used showed their in vitro capacity to differentiate into cardiomyocytes, which expressed troponin T, due to the stimulation of bFGF. Furthermore, they demonstrated their ability to promote angiogenesis through the secretion of VEGF, and monocyte chemoattractant protein-1 (MCP-1), which managed to induce the proliferation of human vascular cells. In conclusion, they indicated that intracoronary administration of autologous BM-MSCs, 1 month after PCI, is tolerable and safe, with a significant improvement in LVEF at 4 and 12 months of follow-up in patients with AMI of the anterior wall.⁸

Lim M et.al, 2018, carried out an experimental study in a porcine model of AMI, forming three groups that were treated with: phosphate buffer saline (PBS); low dose, 0.5 × 10⁶ umbilical cord-derived MSCs (UC-MSCs); and high, 1.5 × 10⁶ UC-MSCs applied intravenously. The doses in the experimental groups were applied 120 minutes and 4 weeks after AMI. In both experimental groups, a trend towards improvement in LVEF was observed at 4 and 8 weeks compared to the control group, but it was not statistically significant. Both groups managed to improve the infarcted area, but did not significantly improve flow at 4 and 8 weeks compared to the control group. Collagen deposition was reduced 3.5 times in the infarcted area, at 8 weeks after AMI in the groups treated with UC MSCs, compared to the control group, which reduced cardiac remodeling. High doses of UC MSCs prevented the downregulation of Cx43 protein in the remote infarct area.⁴

The protein expression of tumor necrosis factor α (TNF α) was lower in the infarcted area and at its border, in the group treated with low doses of UC-MSCs, compared to the control group; In this same group, IL6 expression was also lower at the edge of the infarct. VEGF messenger RNA (mRNA) expression levels were higher at the edge of the infarct in the group treated with high doses of UC MSCs compared to the control group. Platelet/endothelial cell adhesion molecule-1 (PECAM-1; CD31) mRNA was more expressed in the infarcted area and its border in this same group. UC-MSCs were labeled with enhanced green fluorescent protein (eGFP) through a lentivirus transfection process. This methodology allowed the detection of highest expression of the transfected gene on the seventh day in the lung, followed by the kidney, liver, heart, skin and spleen. Furthermore, by immunofluorescence UC-MSCs were detected in the peri-infarcted area; after fourteen days, it was detected that the transfected genes were most expressed in the lung, followed by the spleen, skin, heart, kidney and liver.⁴

Camerlingo C, 2021, carried out an experimental study in an AMI model in rabbits, performed thoracotomies on 5 specimens without inducing AMI and without providing any treatment, 10 animals were induced with AMI and buffered saline solution (PBS) was applied 4 weeks later), and 10 rabbits were administered 10 × 10⁶ ADSCs, derived from epicardium, through injections into the peri-infarct myocardium. The group treated with ADSC presented an average improvement of 4.7%, one month after treatment, in LVEF (95% CI: 5.22, 4.11; p < 0.0001), 0.04 mm reduction in end-diastolic diameter (LVDd) [(95% CI: -0.016, -0.07; p = 0.006)], and 0.08 mm reduction in LVDs [(95% CI: -0.07, -0.097, p = 0.000001)], unlike the control group that presented a decrease of 3.4 % in LVEF (95% CI: -2.63, -4.14, p = 0.000085), 0.07 mm increase in LVDd (95% CI: 0.11, 0.19, p = 0.014), and 0.08 mm increase in LVDs (95% CI: 0.11, 0.047, p = 0.001), it was also observed that the ADSCs transfected with eGFP were located within the infarcted area, after its intramyocardial application and expressed troponin, tropomyosin and desmin, which suggests their differentiation in vivo in cardiomyocytes, these same cells formed new vessels, evidenced by the expression of VEGF.⁵

Kawamura M, 2015, conducted a preclinical study in a porcine model of post-AMI heart disease. After 4 weeks after the infarction was induced, a sternotomy was performed, and 1 × 10⁸ BM human MSCs were applied directly to the infarcted area in the form of 10 cell layers, while in the control group, only the sternotomy was performed. In the subsequent follow-up, a better LVEF was observed in the experimental group, at 4 (51.8% ± 5.4% vs. 35.5% ± 1.9%) and 8 weeks (52.1% ± 3.4% vs. 34.2% ± 4.7%) of treatment. LVDs were lower in the experimental group, 4 weeks (25.6 ± 2.9 mm vs. 29.2 ± 1.3 mm) and 8 weeks (26.1– 2.7 mm vs. 30.3– 1.3 mm) after treatment. Cardiomyocyte hypertrophy, interstitial fibrosis, and vascularity, assessed with PAS staining, Picrosirius red staining, and immunohistochemistry for Von Willebrand factor, respectively, were better in the peri-infarct areas of the experimental group at 8 weeks of follow-up. The diameter of the cardiomyocyte was smaller in the experimental group (13 ± 1 μm vs. 20 ± 2 μm, p < 0.0001), there was less interstitial fibrosis (2.2% ± 0.3% vs. 6.7% ± 1.2%, p < 0.0001), and vascular density was higher (338 ± 59 units/mm2 vs. 139 ± 69 units/mm², p < 0.001) in the same group. After this same period, the expression of the messenger RNA (mRNA) of VEGF and bFGF were higher in the experimental group (VEGF; 0.006 ± 0.004 vs. 0.0005 ± 0.0004, p < 0.05, bFGF; 0.05 ± 0.02 vs. 0.005 ± 0.004, p < 0.01), considering the GAPDH gene as a reference.⁹

Mathiasen A, 2015, conducted a randomized, double-blind, placebo-controlled trial in 60 patients affected by severe ischemic heart failure. He treated 40 patients with BM MSC (77.5±67.9 × 10⁶), applied intramyocardially around the infarcted area, and 20 with placebo. He detected a better LVESV in the group treated with BM MSC, at six months of follow-up, the difference between groups was 13.0±12.9 mL (p < 0.001), the first group also presented better LVEF (6.2±3.8%, p < 0.0001) and better myocardial mass (5.7±7.7 g, p < 0.001). This effect was dose dependent, since the subgroup treated with more than 83x10⁶ MSC BM presented better results in these parameters,¹⁰ and the effects could last up to 4 years.¹¹

Bolli R, 2021, conducted a double-blind, randomized, placebo-controlled trial and applied 108 ± 28 × 10⁶ BM MSC to 22 patients transendocardially. I did not detect significant differences in LVESV, LVEDV and LVEF at 6 and 12 months. of the treatment compared to the placebo group.¹²

Ulus T, 2020, carried out a randomized controlled trial, comparing three groups of patients with chronic ischemic cardiomyopathy (CIC), all treated with coronary artery bypass surgery (CABG), the first only underwent CABG, the second UC was also applied – intramyocardial allogeneic MSCs (21∼26 ×10⁶), and the third additionally autologous bone marrow mononuclear (MN BM), approximately 70 × 10⁷, by the same route. LVEF only improved in the group treated with UC-MSCs, in the magnetic resonance imaging (MRI) evaluation at 12 months of follow-up, in this same group a tendency to gain myocardial mass was observed during the follow-up period, 16 ± 10.6 g, evaluated by MRI. The greatest reduction in necrotic tissue was also noted in this group, 7.7%; as well as greatest distance traveled during 6 minutes, 102.5 ± 43.6 m. In conclusion, they indicated that therapy with UC-MSCs can promote the recovery of patients undergoing coronary revascularization.¹³

Qayyum A, 2013, carried out a multicenter double-blind, placebo-controlled, phase II trial in patients with heart failure due to ischemic causes, in which he applied 100 × 10⁶ ADSCs intramyocardially to 54 patients and saline solution to 27, as controls. At 6 months of follow-up she found no significant differences in LVESV, LVEDV and LVEF.¹⁴

There are many ways to apply stem cells, the doses applied are different and the most opportune moment for their application is controversial, so these characteristics require a thorough evaluation, to promote their correct use and the greatest benefit for the patients with AMI.

Methods

Results

A total of 98 articles were identified in the primary search. After screening titles and abstracts, 11 articles were selected for full-text review. Of these, nine clinical studies were included in the qualitative assessment (see Table 2).

Due to the high heterogeneity of the results with different interventions, doses, a meta-analysis could not be performed.

Discussion

The reviewed studies show significant variability in outcomes based on the MSCs’ administration route and dosage. Intracoronary and intramyocardial administration, particularly at higher doses, tend to yield the most promising results regarding ventricular function improvement and fibrosis reduction. Most studies administered MSCs four weeks post-AMI, likely due to the time required for cell culture.

Two authors used the PCI route and MSCs derived from bone marrow, but only Kim SH et al.⁸ obtained significant clinical results when comparing the experimental group, treated with 7.2 ± 0.9 × 10⁷ BM MSCs, with the control group; while Zhang R et al.,¹ did not detect differences between the group treated with 3.31 ± 1.70 × 10⁶ BM MSCs/4 ml and its control (see Table 1). Given that both authors used the same source of autologous cells, and applied them at similar times, it is likely that the differences are due to the doses used, indicating that a minimum dose of cells is necessary to show a relevant clinical effect, which for The studies described probably correspond to 1 × 10⁶ BM MSCs/kg, if we consider an average weight of 70 kg, to this it is important to add that the in vivo retention percentage of this pathway is 13.74%.⁷ In general, there appear to be common mechanisms of MSCs, which include the secretion of exosomes and the inhibition of the TGF-β/Wnt/Smad signaling pathway,¹⁵ a key pathway in promoting fibrosis in many diseases, including cardiac diseases,¹⁶ that is, they have an antifibrotic effect. Likewise, the pathological replacement process of type I collagen, in the infarcted cardiac tissue, generates the formation of a scar that causes the loss of cardiac contractility, thus the production of HGF and VEGF by the MSCs, reduces fibrosis and promotes vasculogénesis, respectively, as well as the blockade of the TGF-β/Wnt/Smad signaling pathway, must be timely because early inhibition can induce cardiac dysfunction and greater mortality.¹⁷

The intramyocardial route was used by Camerlingo C et al.,⁵ who in an experimental study in rabbits, applied MSCs derived from epicardial adipose tissue, four weeks after AMI was induced, and detected improvements in LVEF of 4.7% (95% CI: 5.22, 4.11, p < 0.0001), 0.04 mm reduction in LVIDd (95% CI: -0.016, -0.07, p=0.006), and 0.08 mm reduction in LVIDs applying a dose of 10 × 10⁶ ADSCs. Another of the authors, who used this route, Mathiasen A et.al, in a double-blind trial, detected differences between the groups in LVESV, of 13.0± 12.9 mL (p < 0.001), LVEF (6.2 ± 3.8%, p < 0.0001) and better myocardial mass (5.7 ± 7.7 g, p < 0.001), using a dose of 77.5 ± 67.9 × 10⁶ of BM MSC,¹⁰ to which we must add that the probable retention of the cells in the myocardium may vary from 14% ± 4%, if applied immediately after the infarction, to 4.5% ± 1.1%, if applied a week later, according to experimental evidence.¹⁸ Considering that both authors used different sources of MSCs, but with positive findings, it is likely that, analogous to the PCI route, the quantity applied is the relevant factor to obtain favorable clinical results; which for the experimental study would correspond to 3 × 10⁶ of ADSCs/kg, considering the average weight of 3 kg of the rabbits; and from 1 to 2 × 10⁶ of BM MSC/kg, in the case of the clinical trial, if we consider an average weight of 70 kg. However, the transdifferentiation capacity of ADSCs, derived from epicardial tissue, is in the experimental study.⁵ In this study, it was possible to verify in vivo the expression of cardiac markers in the applied ADSCs, a fact that, although it has been a source of controversy, has been achieved in vitro, using decellularized matrix of bovine myocardium,¹⁹ without the need for prior genetic modification. in the cells, so it is likely that said transdifferentiation has been promoted by the microenvironment in which the ADSCs were immersed. Transdifferentiation has also been observed in a murine model of AMI treated with UC-MSCs and applied intramyocardially.²⁰

A revolutionary way to treat AMI is through the topical route. Mori D et al.⁶ used 1 × 10⁸ human ADSCs, applying them by spray on the infarcted area, in a porcine model of AMI, which on average was 4 × 10⁸ ADSCs/kg of weight, and detected an improvement in LVEF and LVDs; less fibrosis, less dilation and greater capillarity around the infarcted area. On the other hand, Kawamura M et al.⁹ applied 1 × 10⁸ BM MSCs directly on the epicardium, in the form of cell sheets, at a dose similar to the previous author with similar results. Considering that both authors applied the treatments in comparable situations, approximately one month after AMI, it is evident that a bloody application of the cells is not necessary to obtain favorable results, which enhances the paracrine effect of the MSCs, which is obtained thanks to the release of extracellular vesicles containing pro-angiogenic factors such as VEGF, platelet-derived growth factor (PDGF), angiopoietin, metalloproteases and microRNAs (miR-125a, miR-377), which promote the formation of new vessels. Likewise, molecular messages are transmitted through these vesicles that promote the polarization of monocytes towards type 2 macrophages (anti-inflammatory); and the overexpression of genes such as GATA-4 or Akt, which have an antiapoptotic effect,²¹ all of which favors the recovery of cardiac tissue. Bayes-Genis A et al., (German, 2024) used a similar route to the previous ones, however there were no differences in the groups evaluated, probably because they used a lower dose (7-15 × 10⁶ MSCs).²²

Another route described in the present review was the systemic venous route, which was evaluated by Lim M et.al, 2018, in a porcine model of AMI,⁴ using low and high doses, 0.5 × 10⁶ UC MSCs/Kg and 1.5 × 10⁶ UC MSCs/kg, respectively, with discrete improvement in LVEF and less fibrosis in both groups, lower expression of the inflammatory cytokines TNFα and IL-6, and higher expression of CD31 in the peri-infarct border of the group treated with high doses, but with results quantitatively less forceful than the other pathways, which could be explained by the poor myocardial retention of MSCs, because the cells are retained mainly in the lung.²³ Experimentally, this retention has been estimated at 1.2% ± 0.6%,¹⁸ despite being a safe application route, according to the meta-analysis of Thompson M, 2020,²⁴ it still requires the evaluation of new doses, to rule out, whether its increase could improve the therapeutic effect of this route of application.