Mesenchymal stem cell secretome and exosomes as potential advanced therapy medicinal products for treating a failing heart

Table 1 Pre-clinical studies using mesenchymal stem cell-derived secretome in small and large experimental animal models for cardiac regeneration and repair

Ref.	Model	Source	Isolation methods	Conditioned medium	ROA	Dose and groups	Further details
Timmers et al[59], 2011	Dalland landrace pigs (LCX ligation MI)	hESC-derived MSCs	Centrifugation and 220 nm filtration	Not explicitly mentioned	ICI	MSC-CM, non-CM	CM treatment enhanced cardiac function after MI and suggested potential paracrine-mediated cardioprotection
Hynes et al[67], 2013	Female landrace pigs (LAD occlusion MI)	Porcine EPCs	Centrifuged at 600 × g for 5 minutes + 0.2 μm filtration	IGF-1	ICI	4 mL of X-vivo (control). CM, CM + anti-IGF-1 antibody. CM + Ig. X-vivo + anti-IGF-1 antibody	The data showed IGF-1 as a key mediator of CM’s anti-apoptotic and pro-angiogenic benefits when delivered via ICI
Pavo et al[65], 2014	Porcine (LAD occlusion MI)	Porcine PBMCs (APOSEC)	Dialysis and lyophilization	Not explicitly mentioned	IMI	APOSEC (resuspended in 4 mL physiologic saline, 300 μL aliquots) after 30 days from MI	APOSEC treatment showed long-term improvement in LV pump function, suggesting therapeutic benefits independent of cell differentiation
Vilahur et al[66], 2017	Pigs (LAD ligation MI)	Porcine ASCs	Centrifuged and filtered	GFP	CM: IV. ASCs: ICI (77)	(1) ASCs (1 × 107 cells); (2) CM (30 mL); (3) ASCs + CM; and (4) Control (PBS 30 mL). All of them, 7 days after MI	Explored ASCs and their CM. The ASC-CM group exhibited enhanced micro-vascularization, overcoming ischemia-induced vessel rarefaction
Ellis et al[68], 2021	Mouse exposed to cold UW cardioplegic solution	ASC-S	ASC-S was centrifuged and then filtered using 3 kDa cutoff filters	Antioxidant SOD3, catalase, HGF, VEGF, and SDF-1	I/C infusion of UW ± ASC-S	10% ASC-S in UW solution. Control groups: UW alone or UW + basal media (ASC-BM)	ASC-S significantly improved cardiomyocyte survival, demonstrating its protective role in I/R injury
Könemann et al[64], 2020	Mice (cyclin T1 induced LV-hypertrophy	Murine Sca-1+ & Sca-1- CPCs	0.22 μm filtration + centrifuged for 10 minutes at 300 × g	Not mentioned	IV	Sca-1+ control; Sca-1- control; Sca-1+ Aldo; Sca-1- Aldo, untreated	Sca-1+ CM showed superior cardioprotection, reducing hypertrophy and fibrosis, though both cell types contributed to functional recovery
Huang et al[69], 2020	SD rat (LAD ligation MI) & Yorkshire pigs (LAD ligation MI)	hCSCs	Centrifuged at 1000 × g for 10 minutes	VEGF, HGF, and IGF	TS	artCP (diameter = 3.5 cm patch)	The patch improved myocardial repair by paracrine effects and structural support in rat model. The hCSC-seeded artCP (3.5 cm patch) was implanted via TS. The patch, which releases VEGF, HGF, and IGF, significantly improved cardiac function, demonstrating its scalability for clinical applications

ROA: Route of administration; LCX: Left circumflex; MI: Myocardial infarction; hESC-derived MSCs: Human embryonic stem cell-derived mesenchymal stem cells; ICI: Intra-cardiac injection; CM: Conditioned medium; LAD: Left anterior descending; EPCs: Endothelial progenitor cells; IGF-1: Insulin-like growth factor 1; PBMCs: Peripheral blood mononuclear cells; APOSEC: Apoptotic cell secretome; LV: Left ventricular; ASCs: Adult stem cells; GFP: Green fluorescent protein; IV: Intravenous injection; PBS: Phosphate-buffered saline; UW: University of Wisconsin Solution; ASC-S: Adult stem cell-derived secretome; SOD3: Superoxide dismutase 3; HGF: Hepatocyte growth factor; VEGF: Vascular endothelial growth factor; SDF-1: Stromal cell-derived factor 1; I/C: Intra-corporeal; hCSCs: Human cardiac stem cells; TS: Tissue section; artCP: Articular cardiac patch.

Table 2 Clinical trials evaluating cell-free and cell-based therapies for different cardiovascular diseases

NCT & title	Condition	Intervention	Phase & sample size (n)	Country	Primary outcome parameters	ROA	Dose
Transplantation of MSCs for the prevention of AMI-induced HF. NCT05043610	Prevention of AMI-induced HF	UC-derived WJ-MSCs	Phase 3, n = 390 (completed)	Iran	The primary endpoint for assessing the intervention’s efficacy will be the incidence of HF	I/C	All 130 patients will receive a single intracoronary infusion of 107 WJ-MSCs, alongside the conventional treatment provided to the same number of patients in the control group
Exosome-based Nanoplatform for Ldlr mRNA Delivery in FH (ENDFH). NCT05043181	FH	LDLR mRNA exosomes	Phase 1, n = 30 (not yet recruiting)	China	Safety and preliminary effectiveness of exosome-based LDLR mRNA nanoplatform for gene therapy in HoFH	Abdominal puncture under ultrasound guidance	Escalation phase: Six dose groups are planned (with single doses of 0.044 mg/kg, 0.088 mg/kg, 0.145 mg/kg, 0.220 mg/kg, 0.295 mg/kg, and 0.394 mg/kg, respectively), with three treatments per group. Extension phase: 12 subjects are enrolled and receive three weekly infusions of LDLR mRNA exosomes, with the single dose determined in the dose-escalation phase
Cardiovascular Clinical Project to Evaluate the Regenerative Capacity of CardioCell in Patients with AMI. NCT03404063	AMI	CardioCell	Phase 2/3; n = 105 (completed)	Poland	Improve the clinical outcomes in patients with AMI	Coronary-non-occlusive method (CIRCULATE catheter)	Active IMP consists of 30000000 WJMSCs suspended in 20 mL 0.9% NaCl and 5% albumin administered
Safety and Efficacy of Intracoronary Adult Human Mesenchymal Stem Cells After Acute Myocardial Infarction (SEED-MSC). NCT01392105	AMI	Autologous BM-derived MSCs	Phase 2/3; n = 80 (completed)	South Korea	Safety and efficacy of intracoronary autologous BM-derived hMSCs in patients with AMI	I/C injection	Single dose of autologous BM-derived MSCs. Dosage: 1 × 1000000 cells/kg

NCT: National clinical trial; ROA: Route of administration; MSCs: Mesenchymal stem cells; AMI: Acute myocardial infarction; FH: Familial hypercholesterolemia; UC: Umbilical cord: WJ-MSCs: Wharton’s jelly-derived mesenchymal stem cells; I/C: Intracoronary; LDLR: Low-density lipoprotein receptor; HoFH: Homozygous familial hypercholesterolemia; IMP: Investigational medicinal product; BM: Bone marrow; hMSCs: Human mesenchymal stem cells.

Table 3 Summary of the most commonly employed isolation methods for exosomes with their respective advantages and limitations

Isolation method	Advantages	Limitations	Purity/yield
Differential ultracentrifugation	Well-established and widely used	Labor-intensive; potential co-isolation of proteins	Moderate purity, variable yield
Size-exclusion chromatography	High purity; minimal protein contamination	Requires specialized equipment	High purity, moderate yield
Ultrafiltration	Scalable, GMP-compatible, rapid	Risk of membrane clogging; limited selectivity	Good yield, moderate purity
Polymer precipitation kits	Simple and cost-effective	High protein contamination; unsuitable for clinical-grade use	Low purity, high yield

GMP: Good Manufacturing Practice.

Table 4 Head-to-head comparison of mesenchymal stem cell-based therapy, mesenchymal stem cell-derived exosome-based therapy, and mesenchymal stem cell-derived secretome-based therapy approaches

Parameter	MSC therapy	MSC-derived secretome	MSC-derived exosomes
Composition	Live cells can secrete various bioactive factors	A cell-free mixture containing soluble proteins, cytokines, chemokines, nucleic acids, and small extracellular vesicles released by MSCs	Nanosized vesicles (30-150 nm) enriched with proteins, lipids, and regulatory RNAs (miRNAs) that reflect the molecular profile of their parent MSCs
Isolation and production	Obtained from donor tissue (bone marrow, adipose tissue, or umbilical cord) and expanded under GMP conditions	Produced by culturing MSCs and collecting the conditioned medium, in addition to centrifugation and filtration to remove cells	Generated through further purification of the secretome using ultracentrifugation, filtration, or chromatography
Storage and stability	Requires cryopreservation (-196 °C).	Can be stored frozen (-20 °C to -80 °C) or lyophilized	Similar storage profile to the secretome; stable under freezing or lyophilized conditions
Dosing units	Defined by viable cell number [e.g., (10-100) × 106 cells per dose]	Quantified by total protein concentration or biological activity (e.g., mg of secreted protein)	Expressed by vesicle count (e.g., 1010 particles) or total exosomal protein content (μg)
Mechanism of action	Primarily functions through paracrine signaling, releasing bioactive factors over time and supporting differentiation into cardiac or vascular cells	Acts exclusively through soluble paracrine mediators that promote tissue repair via anti-inflammatory, angiogenic, and regenerative signaling pathways	Delivers specific molecular cargo (miRNAs, proteins, lipids) into target cells, modulating gene expression and promoting tissue regeneration; can be bioengineered for targeted therapeutic effects
Manufacturing complexity	High: Requires GMP-level cell culture, cell banking, karyotype and sterility testing, and cryostorage logistics	Moderate: Requires MSC culture, conditioned medium collection, and downstream processing (e.g., filtration and concentration)	High: Includes all steps of secretome production, specialized purification, and molecular profiling
Scalability	Limited by donor cell proliferation and donor variability. Large-scale expansion increases cost and heterogeneity	Highly scalable; one MSC source can produce large volumes of secretome using culture systems with minimal donor dependence	Partially scalable - while MSC culture is scalable, large-scale exosome isolation and purification remain technically demanding
Cost implications	High production cost due to cell culture, quality control, and cryogenic storage	Lower cost; major expenses arise from culture media and concentration processes, but no live-cell storage is required	Moderate to high cost due to intensive purification techniques

MSC: Mesenchymal stem cell; GMP: Good Manufacturing Practice; miRNAs: MicroRNAs.