Phytocannabinoid-induced priming and differentiation of mesenchymal stem cells: Therapeutic potential

Abstract

Mesenchymal stem cells (MSCs) are multipotent progenitor cells extensively studied for their immunomodulatory and regenerative potential. Despite their therapeutic promise, MSC efficacy can be limited by poor survival, reduced homing, and variable immunoregulatory activity in inflammatory microenvironments. To overcome these challenges, priming strategies have been developed to precondition MSCs, enhancing their functional performance. Among these, phytocannabinoids, bioactive compounds derived from Cannabis sativa, have gained attention due to their ability to modulate MSC behavior. Beyond cannabidiol and Δ⁹-tetrahydrocannabinol, several phytocannabinoids interact with a broad spectrum of receptors, including classical cannabinoid receptors (cannabinoid receptor 1 and cannabinoid receptor 2), G protein-coupled receptor 55, transient receptor potential vanilloid channels, and peroxisome proliferator-activated receptor gamma, influencing intracellular signaling, cytokine expression, migration, viability, and importantly, both MSC priming and lineage differentiation. This mini-review critically examines current in vitro and in vivo evidence on phytocannabinoid-mediated priming and differentiation of MSCs, highlighting their effects on immunomodulation, differentiation, and regenerative potential. Collectively, these findings suggest that phytocannabinoid priming represents a promising approach to enhance MSC therapeutic efficacy, although further studies are required to elucidate receptor-specific mechanisms and optimize priming protocols for clinical translation.

Key Words: Cannabidiol; Cannabinoid receptors; Δ⁹-tetrahydrocannabinol; Endocannabinoid system; Immunomodulation

Core Tip: Phytocannabinoids have emerged as promising modulators of mesenchymal stem cell (MSC) behavior, influencing immunomodulation and differentiation. Although a few in vivo studies exist, the majority of evidence comes from in vitro models. However, challenges remain for the clinical application of MSC priming with phytocannabinoids or the secretome derived from primed cells. Standardized priming protocols, deeper mechanistic insights, and comprehensive in vivo validation are crucial to fully harness their therapeutic potential.

INTRODUCTION

Cell-based therapies employing mesenchymal stem cells (MSCs) have emerged as a promising strategy for treating a wide range of diseases, owing to their potent anti-inflammatory, immunomodulatory, and anti-apoptotic properties[1]. The therapeutic effects of MSCs are largely attributed to their paracrine activity, which involves the secretion of soluble factors and extracellular vesicles, as well as direct cell-to-cell interactions that influence immune and tissue-resident cells[2]. Through these mechanisms, MSCs modulate the activity of T lymphocytes, natural killer cells, neutrophils, and macrophages, thereby orchestrating an immunoregulatory environment conducive to tissue repair[3].

However, despite their recognized therapeutic potential, the survival, engraftment, and functional stability of MSCs following transplantation may often be compromised by the hostile microenvironment of injured tissues[4]. Conditions including oxidative stress and inflammation may challenge MSC survival, reducing therapeutic efficacy[5]. To overcome these limitations, various priming approaches have been explored. This strategy involves exposing MSCs to controlled stimuli, such as inflammatory cytokines [e.g., tumor necrosis factor-α (TNF-α), interferon-γ], hypoxic conditions, or pharmacological agents, to enhance their paracrine secretion, immunomodulatory profile, and regenerative performance[6].

Among priming agents, phytocannabinoids [e.g., cannabidiol (CBD) and Δ⁹-tetrahydrocannabinol (THC)] have gained increasing attention. These compounds may modulate the immunoregulatory activity of MSCs through multiple molecular pathways, including activation of cannabinoid receptor type 2 (CB2) receptors and extracellular signal-regulated kinase signaling, as well as suppressing NALP3 inflammasome activation[7,8]. In addition, they have been reported to affect MSC differentiation into specific lineages, suggesting a role in shaping cell fate[9]. Thus, this mini-review aims to summarize and critically analyze current evidence on the priming and differentiation of MSCs induced by phytocannabinoids, highlighting their emerging roles in immunomodulation and their prospective contribution to the development of MSC-based therapeutic applications.

SEARCH STRATEGY

A comprehensive literature search was performed using the PubMed database to identify relevant studies published up to October 30, 2025. The search strategy included the following keywords and their combinations: “mesenchymal stem cells”, “phytocannabinoid”, “Δ⁹-tetrahydrocannabinol”, “cannabidiol”, “endocannabinoid system”, “priming”, “preconditioning”, “pre-treatment”, “cannabigerol”, “cannabinol”, and “cannabichromene”. Only articles in English were considered.

The initial search included studies published from 2015 to 2025 to encompass the most recent advances in the field. A subsequent comprehensive search, without time restrictions, was then conducted to ensure full coverage of relevant literature. Titles and abstracts were screened by the authors, and full texts were consulted when necessary. The review was conducted by two authors, and no formal conflict resolution process was applied. Studies were deemed eligible if they evaluated the effects of phytocannabinoids on MSCs, including aspects such as priming, immunomodulatory activity, and differentiation potential. Both in vitro and in vivo experiments were considered, while those not directly addressing MSC priming or differentiation were excluded.

MSCS

MSCs are multipotent, non-hematopoietic cells that can be isolated from a variety of adult tissues[10]. These cells exhibit broad differentiation potential across multiple lineages, which makes them promising candidates for therapeutic applications[11]. MSCs have been obtained from sources such as bone marrow, adipose tissue, umbilical cord, placenta, dental pulp, and synovial membrane[12]. The International Society for Cellular Therapy has established minimal criteria for the characterization of human MSCs. These criteria include adherence to plastic under standard culture conditions, a fibroblast-like morphology, expression of specific surface markers (CD73, CD90, and CD105), absence of hematopoietic and endothelial markers (CD14 or CD11b, CD34, CD45, CD79α or CD19, and HLA-DR), and the capacity to differentiate into chondrogenic, osteogenic, and adipogenic lineages[13]. In addition to their differentiation potential, MSCs exert crucial effects, primarily through their paracrine activity, by releasing soluble factors and extracellular vesicles[14]. These secreted components contribute to immunomodulation, anti-inflammatory effects, neuroprotection, angiogenesis, tissue repair, and antiapoptotic activity[15-17].

Interestingly, MSCs also secrete endocannabinoids, such as anandamide (AEA) and 2-arachidonoylglycerol (2-AG), which contribute to the multifaceted therapeutic potential of MSCs[18] (Figure 1). Although MSCs have considerable therapeutic potential, their survival and functional performance after transplantation can be compromised by unfavorable microenvironmental factors, such as inflammatory signaling, oxidative stress, and limited nutrient availability, leading to poor engraftment and diminished therapeutic outcomes[19,20]. These challenges have motivated the development of strategies aimed at enhancing the therapeutic efficacy of MSCs.

Figure 1 Schematic representation of the paracrine effects of mesenchymal stem cells. Mesenchymal stem cells exert therapeutic effects primarily through the paracrine release of soluble factors and extracellular vesicles. They modulate immune responses by secreting interleukin-10 (IL-10), IL-6, indoleamine 2,3-dioxygenase, prostaglandin E₂, hepatocyte growth factor (HGF), transforming growth factor-β, and nitric oxide; promote angiogenesis via vascular endothelial growth factor, insulin-like growth factor-1 (IGF-1), IL-6, monocyte chemoattractant protein-1, angiopoietin-1, and angiogenin; and prevent apoptosis through HGF, IGF-1, vascular endothelial growth factor, and transforming growth factor-β. In addition, neuroprotective molecules such as IGF-1, HGF, nerve growth factor, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, fibroblast growth factor-2, and ciliary neurotrophic factor support neuronal survival and regeneration. Mesenchymal stem cells also release bioactive lipids, including 2-arachidonoylglycerol and anandamide, which contribute to their therapeutic potential[18]. Created in BioRender. Amorim, R. (2026) https://BioRender.com/0lqgoi6. IL: Interleukin; IDO: Indoleamine 2,3-dioxygenase; PGE₂: Prostaglandin E₂; HGF: Hepatocyte growth factor; TGF-β: Transforming growth factor-β; NO: Nitric oxide; 2AG: 2-arachidonoylglycerol; AEA: Anandamide; VEGF: Vascular endothelial growth factor; IGF-1: Insulin-like growth factor-1; MCP-1: Monocyte chemoattractant protein-1; MSCs: Mesenchymal stem cells; BDNF: Brain-derived neurotrophic factor; GDNF: Glial cell line-derived neurotrophic factor; FGF-2: Fibroblast growth factor-2; CNTF: Ciliary neurotrophic factor.

THE ENDOCANNABINOID SYSTEM AND PHYTOCANNABINOIDS

The endocannabinoid system (ECS) is a conserved signaling network found throughout the phylum Chordata and in invertebrates such as Porifera, Cnidaria, Nematoda, and Arthropoda, highlighting its evolutionary relevance[21]. The classic ECS comprises cannabinoid receptors, endogenous ligands (endocannabinoids), and enzymes responsible for their synthesis, transport, and degradation. Collectively, these components regulate diverse physiological functions within the nervous, cardiovascular, reproductive, and immune systems[22].

The concept of an expanded ECS, termed the endocannabinoidome, extends beyond the classical CB1 and CB2 receptors to encompass a broad array of receptor-ligand interactions and signaling networks[23] (Figure 2). Collectively, these molecular targets orchestrate diverse physiological processes, such as energy metabolism, inflammation, nociception, and cellular differentiation. This complexity reflects the dynamic influence of the cellular microenvironment, including lipid mediator availability, receptor conformations, and ligand-induced signaling, which collectively shape the biological outcomes of cannabinoid signaling[24].

Figure 2 Schematic overview of cannabinoid receptor-mediated signaling and phytocannabinoid-induced modulation and differentiation of mesenchymal stem cells. Phytocannabinoids such as cannabidiol (CBD) and Δ⁹-tetrahydrocannabinol interact with multiple molecular targets expressed in mesenchymal stem cells (MSCs), including cannabinoid receptors type 1 (CB1) and type 2 (CB2). Endogenous cannabinoids such as anandamide activate both CB1 and CB2, while CBD inhibits fatty acid amide hydrolase, preventing anandamide degradation and consequently enhancing endocannabinoid receptor signaling. Activation of CB2 predominantly engages the p38 and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (p42/44) pathways, promoting osteogenic differentiation and MSC migration, whereas CB1 activation modulates neuronal differentiation. Additionally, combined treatment with moringin and CBD has been shown to enhance MSC survival and promote neuronal differentiation through activation of the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of the rapamycin signaling pathway. In turn, activation of peroxisome proliferator-activated receptor gamma promotes adipogenic differentiation. These receptor-mediated cascades converge to regulate not only lineage commitment but also the paracrine activity of MSCs, leading to the release of extracellular vesicles and bioactive molecules. Created in BioRender. Amorim, R. (2026) https://BioRender.com/0lqgoi6. PPARγ: Peroxisome proliferator activated receptor gamma; ERK: Extracellular signal-regulated kinase; MAPK: Mitogen-activated protein kinase; PI3K: Phosphatidylinositol 3-kinase; Akt: Protein kinase B; mTOR: Mammalian target of the rapamycin; CB2: Cannabinoid receptor type 2; GPR55: G protein-coupled receptor 55; CB1: Cannabinoid receptor type 1; CBD: Cannabidiol; THC: Δ⁹-tetrahydrocannabinol; AEA: Anandamide; MOR: Moringin; FAAH: Fatty acid amide hydrolase.

Cannabinoid receptors belong to the G protein-coupled receptor family. CB1 is predominantly expressed in the central and peripheral nervous systems, where it modulates neurotransmitter release and neuronal excitability, whereas CB2 is primarily found in immune cells and mediates cytokine secretion, chemotaxis, and inflammatory responses[25,26]. Importantly, both receptors have also been expressed in MSCs from different species, including human[27], mouse[8], and equine[28], suggesting a conserved regulatory role in MSC biology.

Both CB1 and CB2 activate intracellular pathways, including mitogen-activated protein kinase (MAPK) and adenylyl cyclase, primarily via heterotrimeric G proteins, with β-arrestins contributing to MAPK signaling and receptor regulation[29]. CB1 activation has been shown to promote neuronal differentiation and maturation of neural stem cells[30], whereas CB2 activation enhances osteogenic differentiation and cell migration[31,32].

Among the more than 120 phytocannabinoids identified in Cannabis sativa, THC and CBD are the most extensively studied and pharmacologically relevant compounds[33,34]. Other phytocannabinoids such as cannabigerol (CBG), cannabinol (CBN), and cannabichromene (CBC) have shown emerging biological activities[35], but their mechanisms in MSCs remain poorly characterized. THC acts as a partial agonist at both CB1 and CB2, mediating analgesic and anti-inflammatory effects[36,37]. A previous study reported that THC enhances MSC migration through CB1 receptor-dependent activation of p42/44 MAPK, as evidenced by the significant inhibition of both MAPK phosphorylation and migratory responses following pharmacological blockade with the CB1 antagonist AM251[38].

CBD acts on several receptor systems, including CB1, CB2, G protein-coupled receptor 55 (GPR55), transient receptor potential vanilloid channels (TRPV), and peroxisome proliferator-activated receptor gamma (PPARγ), thereby mediating neuroprotective, anti-inflammatory, analgesic, anxiolytic, and antitumor effects[39]. Moreover, a previous study demonstrated that fatty acid amide hydrolase (FAAH) inhibitors and substrates induce p42/44 MAPK phosphorylation, which subsequently activates PPARγ and enhances MSC migration[40]. In addition, CBD has been shown to promote MSC migration through CB2 receptor activation and functional inhibition of GPR55 signaling, as evidenced by the suppression of CBD-induced migration following treatment with the CB2 antagonist AM-630 and the GPR55 agonist O-1602[31].

The expression of cannabinoid receptors in MSCs and their responsiveness to phytocannabinoids indicate a functional interplay between the ECS and stem cell physiology. This interaction underlies the rationale for exploring phytocannabinoid-based priming strategies, aiming to enhance the immunomodulatory, regenerative, and differentiation capacities of MSCs.

PHYTOCANNABINOID-BASED PRIMING OF MSCS

Given the limitations associated with MSC-based therapies, priming of MSCs has emerged as a strategy to enhance their therapeutic potential[41]. Among the various priming approaches, exposure to phytocannabinoids has shown promise in promoting immunomodulatory responses in MSCs[7,28,42,43]. Interestingly, MSCs are naturally responsive to cannabinoid signaling, as they secrete and respond to endogenous cannabinoids such as AEA and 2-AG. These endocannabinoids exert immunosuppressive effects on both macrophages and MSCs[44]. In macrophages stimulated with lipopolysaccharide (LPS), AEA reduced interleukin (IL)-6 and TNF-α secretion, while 2-AG decreased IL-6, macrophage migration inhibitory factor (MIF), and TNF-α levels. In MSCs, both compounds reduced MIF expression, supporting their potential as therapeutic agents against inflammatory disorders[44]. Notably, CBD can indirectly enhance endocannabinoid signaling by inhibiting FAAH, the enzyme responsible for AEA degradation[45]. This functional interaction between phytocannabinoids and the endogenous cannabinoid system provides a biological rationale for exploring phytocannabinoid-based priming as a strategy to enhance the immunomodulatory properties of MSCs.

Consistent with this concept, studies have demonstrated that priming of MSCs with cannabinoids such as CBD or THC can modulate key signaling pathways and cytokine expression profiles (Table 1). These effects depend on several factors, including the form and purity of the phytocannabinoid, its concentration, and the presence or absence of inflammatory stimuli during treatment. Several studies have explored the priming of MSCs with phytocannabinoids in combination with inflammatory stimuli such as LPS or TNF-α, demonstrating that this can promote immunomodulatory responses in MSCs[8,34]. Although these findings are promising, they are largely limited to in vitro conditions. Clinically, MSCs are often transplanted into inflamed or hostile tissues, raising the important translational question of whether phytocannabinoid pre-treatment could enhance MSC survival, immunomodulatory function, and functional integration in such environments. LPS and TNF-α create an inflammatory environment in vitro, attempting to mimic the hostile conditions MSCs would encounter in vivo. Thus, future studies should directly test these effects in animal models to determine the efficacy of priming under clinically relevant conditions.

Table 1 Experimental conditions and outcomes of phytocannabinoid-based priming in mesenchymal stem cells.

Cell	Phytocannabinoid	Period of priming	Stress/inflammatory condition	Dose	Effect	Notes	Limitation	Ref.
GMSCs (human)	Pure CBD (> 99%)	24 hours	No	5 μM	Downregulated the expression of genes IL6ST, IL-1β, and IL-18	CBD modulated the expression of 5843 genes in GMSCs and downregulated the expression of genes correlated to inflammation, apoptosis and innate immune responses. CBD attenuated activation of the NLRP3 inflammasome, accompanied by a decline in NLRP3, CASP1, and IL-18 expression	No in vivo evaluation	[7]
AT-MSCs (human)	CBD (not specified)	48 hours	LPS (10 μg/mL)	3 μM	Multiplex immunoassay and ELISA showed that CBD + LPS did not alter the release of cytokines and growth factors (IL-2, IL-5, IL-6, IL-18, TNF-α, TGF-β, VEGF, IGF) compared to the LPS group	An increase in IL-6 and VEGF levels and a decrease in IGF were observed in the CBD group compared to the unstimulated control group	Receptors mediating the observed effects were not investigated. No in vivo evaluation	[34]
AT-MSCs (human)	CBD (1089161 - Sigma-Aldrich)	24 hours	Tunicamycin	5 μM	Decreased IL-4 and increased gene and protein expression of IL-6 compared with the tunicamycin-treated group. CBD significantly reduced the number of senescent cells	Increased gene expression of IL-1β, IL-4, and IL-6, and decreased expression of TNF-α and IL-10 compared with the control group. CBD reduced apoptosis and promoted proliferation in pretreated cells	Receptors mediating the observed effects were not investigated. No in vivo evaluation	[42]
DPMSCs (human)	CBD (not specified)	24 hours	TNF-α (50 ng/mL)	2.5 μM	CBD treatment reduced TNF-α-induced gene expression of TNF-α, IL-6, and IL-1β	CBD treatment upregulated the gene expression of pro-angiogenic markers and restored TNF-α-inhibited viability and migration	Absence of cytokine protein analysis. Receptors mediating the observed effects were not investigated. No in vivo evaluation	[47]
AT-MSCs (canine)	CBD-rich cannabis extract (28.12% CBD and 0.8% THC)	24 hours	No	2.25 μM and 225 nM	Gene expression analysis showed decreased BDNF (2.25 μM), increased HGF (2.25 μM and 225 nM), and increased IDO (2.25 μM). Multiplex assay revealed a reduction in IL-8 and MCP-1 levels (2.25 μM)	No significant differences were observed in the gene expression of GDNF, IL-10, TNF-α, IFN-γ, or PTGES2 compared to the control. CBD did not alter cell morphology and viability	Lack of protein-level analysis of additional cytokines and neurotrophic factors. Receptors mediating the observed effects were not investigated. No in vivo evaluation	[43]
AT-MSCs (equine)	CBD-rich cannabis extract (28.12% CBD and 0.8% THC)	24 hours	No	5 μM and 7 μM	Gene expression analysis showed decreased IL-1β and IL-6 (7 μM) and increased IL-10, IFN-γ, and TNF-α (5 μM)	No significant changes were observed at 5 μM for IL-1β and IL-6, or at 7 μM for IL-10, IFN-γ, and TNF-α compared with the control. CBD did not alter cell morphology, viability, metabolic activity, or β-galactosidase activity	Absence of cytokine protein analysis. No in vivo evaluation	[28]
BM-MSCs (mice)	THC (Sigma Aldrich)	24 hours	LPS-stimulated microglia (in vitro)/CCI model in mice (in vivo)	1 μM (chosen after testing 0.5-10 μM)	Enhanced immunomodulatory effect: Lowered release of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8) from microglia; increased IL-10; improved outcomes in pain behavior (hyperalgesia, allodynia) in CCI mice; reduced cytokine expression in ipsilateral sciatic nerve	Mechanism involves CB2 receptor; ERK and Akt signaling pathways	Long-term or dose-dependent effects not evaluated	[8]

GMSCs: Gingival mesenchymal stem cells; CBD: Cannabidiol; IL: Interleukin; NLRP3: Nucleotide-binding oligomerization domain-, leucine-rich repeat-, and pyrin domain- containing receptor 3; CASP1: Caspase-1; AT-MSCs: Adipose tissue-derived mesenchymal stem cells; LPS: Lipopolysaccharide; ELISA: Enzyme-linked immunosorbent assay; TNF-α: Tumor necrosis factor-alpha; TGF-β: Transforming growth factor β; VEGF: Vascular endothelial growth factor; IGF: Insulin-like growth factor; DPMSCs: Dental pulp mesenchymal stem cells; THC: Δ⁹-tetrahydrocannabinol; BDNF: Brain-derived neurotrophic factor; HGF: Hepatocyte growth factor; IDO: Indoleamine 2,3-dioxygenase; MCP-1: Monocyte chemoattractant protein-1; GDNF: Glial cell line-derived neurotrophic factor; IFN-γ: Interferon-gamma; PTGES2: Prostaglandin E synthase 2; BM-MSCs: Bone marrow-derived mesenchymal stem cells; CCI: Chronic constriction injury; CB2: Cannabinoid receptor type 2; ERK: Extracellular signal-regulated kinase; Akt: Protein kinase B.

It is important to note that most studies employ CBD concentrations below 10 μM, as higher doses have been associated with reduced MSC viability at 10 μM[46], decreased cell proliferation at 12.5 μM[47], and cell death at 10 μM and 25 μM[48]. However, such effects may vary according to the phytocannabinoid source, formulation, and priming duration. Results consistently indicate that CBD or THC exposure can regulate cytokine expression (e.g., TNF-α, IL-6, IL-1β, IL-10), modulate immune-related genes, and, in some cases, enhance angiogenic or neuroprotective features[28,43,47].

In line with these findings, in a murine excisional wound-healing model, the genetic deletion of CB1 or CB2 receptors revealed distinct roles of the ECS in inflammation and tissue repair. Deletion of CB2 increased IL-6 and TNF-α levels in wound tissue but did not affect the overall regenerative process[49]. In contrast, CB1-deficient mice exhibited delayed wound closure during the early healing phase, accompanied by elevated concentrations of monocyte chemoattractant protein-1 (MCP-1) and TNF-α[49]. Adipose tissue-derived MSCs isolated from CB1^-/- and CB2^-/- animals displayed altered viability and differentiation capacity compared to wild-type MSCs. Notably, CB1^-/- MSCs secreted higher levels of MCP-1 upon TNF-α and IL-1β stimulation, suggesting that the loss of CB1 signaling disrupts the MSC secretome and contributes to delayed wound repair[49].

In addition, low-dose THC administration improved wound healing in aged mice by accelerating closure and reducing inflammatory cytokines, while also increasing MSC infiltration into the wound tissue[50]. However, several factors require careful consideration. Some studies have employed phytocannabinoid-rich extracts rather than purified compounds, which introduces variability in composition and potency. The chemical profile of such extracts can differ markedly depending on the plant’s geographical origin, cultivation conditions, and regulatory standards, ultimately influencing the relative concentrations of CBD, THC, and other bioactive constituents. Likewise, variations in priming parameters, such as duration and concentration, can impact the immunomodulatory activity of MSCs. These methodological inconsistencies likely contribute to the divergent cytokine profiles reported among studies, underscoring the need for standardized priming protocols to ensure reproducible and comparable outcomes.

Despite promising findings, most available studies are limited to in vitro conditions and short-term priming, with scarce evidence regarding receptor-specific mechanisms or long-term functional outcomes in vivo. Further research is warranted to define optimal priming conditions, elucidate cannabinoid receptor involvement (CB1, CB2, or others), and validate the translational relevance of cannabinoid-primed MSCs in regenerative medicine. Moreover, increasing attention should be given to the secretome derived from phytocannabinoid-primed MSCs, which, to the best of our knowledge, has not been evaluated in vivo and may represent a safer and more controllable therapeutic approach by harnessing the paracrine effects of priming without requiring direct cell transplantation.

PHYTOCANNABINOID-MEDIATED DIFFERENTIATION OF MSCS

Phytocannabinoids have emerged as bioactive modulators of MSC fate, influencing lineage specification and functional phenotype through complex interactions with the ECS. The ability to direct MSC differentiation toward specific lineages holds therapeutic relevance for regenerative medicine, as it may enhance tissue repair or replacement by generating functionally specialized cells.

Evidence indicates that phytocannabinoids can modulate MSC differentiation in different lineages (Table 2)[31,46-48,51-59]. Activation of the CB2 receptor has been shown to enhance osteogenic differentiation of human bone marrow-derived MSCs, resulting in increased alkaline phosphatase activity, calcium deposition, and upregulation of osteogenic genes[32]. In addition, CBD has been reported to promote osteogenic differentiation of mouse bone marrow-derived MSCs under inflammatory conditions via CB2 receptor activation and subsequent engagement of the p38 MAPK signaling pathway[51]. This effect was confirmed by the loss of osteogenic benefits following treatment with the CB2 antagonist AM630 and the selective p38 inhibitor SB530689[51].

Table 2 Experimental conditions and outcomes of phytocannabinoid-based differentiation of mesenchymal stem cells.

Cell	Phytocannabinoid	Period	Stress/inflammatory condition	Dose	Differentiation	Notes	Limitation	Ref.
BM-MSCs (human)	VCE-003.2 - a quinone derivate of CBG	14 days for gene expression and 21 days for staining	No	1 μM and 2.5 μM	Adipogenic - adipogenic differentiation medium in the presence of VCE-003.2 enhanced the expression of adipogenic markers such as PPARγ, LPL, CEBPA, ADIPOQ, and FABP4. Osteogenic - VCE-003.2 did not interfere with osteoblastic differentiation	The percentage of Oil Red O-positive cells induced by VCE-003.2 was lower than that obtained with rosiglitazone	No staining confirmation of osteogenic differentiation was performed. Differentiation was not assessed under inflammatory or stress conditions	[54]
BM-MSCs (human)	VCE-003.2 - a quinone derivate of CBG	7 days	No	1 μM	Adipogenic - increased expression of adipogenic-related genes PPARγ2, FABP4, ADIPOQ, LPL, and CEBPα	VCE-003.2 activates the nuclear receptor PPARγ	No staining confirmation of differentiation. Differentiation not assessed under inflammatory/stress conditions	[55]
BM-MSCs (mouse)	CBD, CBG, CBGA, and THCV (GW Research Ltd)	14 days	No	1 μM, 3 μM, and 5 μM	Adipogenic - CBG (5 μM) and CBD (5 μM) alone or in combination promote MSCs maturation into adipocytes. Increase of Fabp4 gene expression (5 μM CBD)	CBD, CBDA, CBGA, and THCV (5 μM) increase BM-MSC viability. CBDA, CBG, or CBD, alone or in combination, modulate multiple key receptors, including CB2 and PPARγ	Differentiation not assessed under inflammatory/stress conditions	[56]
BM-MSCs (human and mouse)	99% pure CBD crystals (CBDistillery)	14 days for murine MSCs and 21 days for human MSCs	No	2.5 μM, 5 μM, and 10 μM	Adipogenic - in hMSCs, CBD increased expression of adipogenic genes PPARγ2, FABP4, and FSP27. In mMSCs, all doses upregulated PPARγ2, whereas FABP4 and FSP27 responded only at 10 μM. Nile Red staining showed that CBD induced a dose-dependent increase in lipid accumulation in hMSCs, whereas in mMSCs only 5 μM and 10 μM promoted lipid deposition, with no effect observed at 2.5 μM	CBD induced adipogenic differentiation in MSCs through a PPARγ-dependent mechanism. CBD exhibited a stronger adipogenic effect in hMSCs compared to mMSCs	Differentiation not assessed under inflammatory/stress conditions	[52]
MSCs (human) source not specified	HSO, CBD, THC	72 hours	No	HSO: 0.1% and 0.05%. CBD: 1 μM. THC: 1 μM	HSO, CBD, and THC initiated adipogenic differentiation in hMSCs with or without DM. RT-qPCR analysis of PPARγ and CEBPα showed: In DM, THC increased PPARγ, 0.1% HSO decreased PPARγ; CEBPα mRNA was generally increased by treatments in DM except THC, while all treatments decreased CEBPα in treatment-only groups	HSO downregulated CB1 mRNA and protein expression of the endogenous ligand TRPV1; endogenous CB1 inhibition neutralized or reduced FAAH and MGL expression. While HSO alone can initiate adipogenic differentiation and upregulate PPARγ in hMSCs, it inhibits CB1, TRPV1, and PPARγ under DM conditions	No in vivo studies to validate CB1 inhibition or assess complete adipocyte maturation	[57]
AT-MSCs (human)	CBD (Farmech Società Agricola SRL, Italy)	3 days, 7 days, and 14 days for gene expression; 14 days and 21 days for staining	No	0.1 μM, 0.5 μM, 2.5 μM and 5 μM	Adipogenic - spontaneous formation of lipid vacuoles in their cytoplasm. Adipogenesis significantly increased with 2.5 μM and 5 μM compared to untreated. Gene expression of PPARγ and CEBPα was upregulated from day 3, and FABP4 from day 7 (5 μM)	Gene expression of osteogenic markers RUNX2 significantly decreased at day 14, and COL1A1 was downregulated from day 3	Differentiation not assessed under inflammatory/stress conditions. Mechanism/pathway not assessed	[46]
AT-MSCs (human)	CBD (R&D Systems)	3 days and 14 days for gene expression analysis, and 28 days and 35 days for mineralization assessment	No	3 μM	Osteogenic - increase of ALP activity and mineralization. Upregulation of osteogenic genes BMP-2, BMP-7, CSF2, MSX-1, and ENAM (14 days)	CBD enhances MSC migration through CB2 receptor activation and GPR55 inhibition	Differentiation not assessed under inflammatory/stress conditions	[31]
DPMSCs, DFMSCs, and APMSCs (human)	CBD and vitamin D3 (not specified)	10 days for gene expression and 21 days for staining	No	CBD (0.75 μM, 0.5 μM) + vitamin D3 (10 nM and 5 nM) for staining and CBD (0.75 μM) + vitamin D3 (2.5 nM) for gene expression	Osteogenic - CBD enhanced osteogenic gene expression (COL1A1, osteopontin, OCN, osteonectin)	DPMSCs: Best osteogenic response to vitamin D3; APMSCs - highest bone gene expression with low-dose CBD; DFMSCs: Strongest mineralization with CBD and vitamin D3	Differentiation not assessed under inflammatory/stress conditions. Mechanism/pathway not assessed	[58]
BM-MSCs (mouse)	CBD (CATO, United States)	7 days and 14 days	LPS (10 μg/mL)	0.5 μM, 2.5 μM and 5 μM	Osteogenic - increased gene and protein expression of RUNX2, ALP, and OCN after 7 days. CBD also enhanced calcium nodule deposition, as shown by Alizarin Red staining after 14 days	Enhanced osteogenic differentiation of MSCs, partly via p38 MAPK signaling	CB2 inhibition did not fully block CBD-induced osteogenesis, suggesting additional mechanisms	[51]
SMSCs (human)	CBD (CBDistillery)	4 days	No	2 μg/mL	Osteogenic - increase of gene expression of OCN	No changes in gene expression of RUNX2 and osterix. CBD increased cell viability and proliferation	Mechanism/pathway not assessed. No staining confirmation of differentiation. Differentiation not assessed under inflammatory/stress conditions in vitro	[59]
DPMSCs (human)	CBD (not specified)	4 days, 7 days, 14 days	TNF-α (20 ng/mL and 50 ng/mL)	0.1 μM, 0.5 μM, 2.5 μM	Osteogenic - CBD promoted the ALP staining of DPMSCs at 0.1-2.5 μM concentrations. After 14 days of induction, alizarin red staining showed enhanced matrix mineralization in CBD-induced group. 2.5 μM CBD upregulated mRNA expression of ALP, osteopontin, OCN, osteonectin, and COLI	Among the concentration tested, 2.5 μM CBD exerted the highest effect on the proliferation and osteogenic differentiation of DPMSCs	Mechanism/pathway not assessed	[47]
GMSCs (human)	CBD (> 99% pure) - (Carmagnola, Italy)	24 hours for gene expression and 48 hours and 96 hours for immunocytochemistry	No	5 μM, 10 μM and 25 μM	Neurogenic - MSCs with 5 μM CBD upregulated CES1, CHRM2, and DPCR1. After 48 hours of treatment, TP53 protein expression was detected, whereas NEFM and CHRM2 were not expressed. At 96 hours, this pattern was reversed, with positive expression of NEFM and CHRM2	CBD at 10 μM and 25 μM induced cell death	Differentiation not assessed under inflammatory/stress conditions	[48]
PL-MSCs (human)	Pure CBD (> 99%) + MOR	48 hours	No	CBD and MOR (1:1 mixture, 0.5 μM)	Neurogenic - increased protein expression of nestin, GAP43, GFAP, and BDNF	CBD improved MSC survival; prolonged survival associated with apoptosis inhibition via PI3K/Akt/mTOR pathway	Groups with only CBD or only MOR not included; cellular functionality not assessed	[53]

BM-MSCs: Bone marrow-derived mesenchymal stem cells; CBG: Cannabigerol; PPARγ: Peroxisome proliferator activated receptor gamma; LPL: Lipoprotein lipase; CEBPα: CCAAT/enhancer-binding protein alpha; ADIPOQ: Adiponectin; FABP4: Fatty acid-binding protein 4; FSP27: Fat-specific protein 27; CBD: Cannabidiol; CBGA: Cannabigerolic acid; MSCs: Mesenchymal stem cells; CBDA: Cannabidiolic acid; THCV: Tetrahydrocannabivarin; CB2: Cannabinoid receptor 2; hMSCs: Human mesenchymal stem cells; mMSCs: Mouse mesenchymal stem cells; HSO: Hemp seed oil; THC: Δ⁹-tetrahydrocannabinol; DM: Differentiation medium; CB1: Cannabinoid receptor 1; TRPV1: Transient receptor potential vanilloid 1; FAAH: Fatty acid amide hydrolase; MGL: Monoacylglycerol lipase; AT-MSCs: Adipose tissue-derived mesenchymal stem cells; RUNX2: Runt-related transcription factor 2; COL1A1: Collagen type I alpha 1 chain; ALP: Alkaline phosphatase; BMP-2: Bone morphogenic protein 2; BMP-7: Bone morphogenic protein 7; CSF2: Colony-stimulating factor 2; MSX-1: MSH homeobox 1; ENAM: Enamelin; GPR55: G protein-coupled receptor 55; DPMSCs: Dental pulp mesenchymal stem cells; DFMSCs: Dental follicle-derived mesenchymal stem cells; APMSCs: Apical papilla-derived mesenchymal stem cells; OCN: Osteocalcin; LPS: Lipopolysaccharide; MAPK: Mitogen-activated protein kinase; SMSCs: Skeletal mesenchymal stem cells; TNF-α: Tumor necrosis factor alpha; COL-I: Collagen type I; GMSCs: Gingival mesenchymal stem cells; CES1: Carboxylesterase 1; CHRM2: Cholinergic receptor muscarinic 2; DPCR1: Diffuse panbronchiolitis critical region 1; TP53: Tumor protein p53; NEFM: Neurofilament medium polypeptide; PL-MSCs: Periodontal ligament mesenchymal stem cells; MOR: Moringin; GAP43: Growth-associated protein 43; GFAP: Glial fibrillary acidic protein; BDNF: Brain-derived neurotrophic factor; PI3K: Phosphatidylinositol 3-kinase; Akt: Protein kinase B; mTOR: Mammalian target of the rapamycin.

Additional in vitro and in vivo findings indicate that CBD can enhance osteogenic effects. In a rat model of bone injury, CBD treatment increased the regenerative capacity of a scaffold, which was associated with enhanced recruitment of MSCs and upregulation of osteogenesis-related genes[60]. CBD has also been shown to induce adipogenic differentiation of MSCs through PPARγ activation, leading to lipid accumulation and increased expression of adipogenic markers such as CCAAT/enhancer-binding protein alpha and fatty acid-binding protein 4[46,52]. This was mechanistically substantiated by the significant attenuation of differentiation upon administration of the selective PPARγ antagonist T0070907[52]. In human adipose-derived MSCs, LPS inhibited both adipogenic and chondrogenic differentiation, which was mitigated by CBD treatment[34].

Furthermore, one study showed that treatment with a combination of moringin and CBD improved the survival and neuronal differentiation potential of human periodontal ligament MSCs[53]. This effect was associated with activation of the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of the rapamycin signaling pathway and increased expression of neuronal markers such as nestin and growth associated protein 43[53] (Figure 2). However, it remains unexplored whether CBD alone can trigger neuronal lineage differentiation. Collectively, current evidence indicates that phytocannabinoids can modulate MSC differentiation. By engaging the ECS and interacting with key intracellular signaling pathways, these compounds can bias MSC fate toward osteogenic, adipogenic, and potentially neuronal lineages. However, the differentiation outcome is likely influenced by microenvironmental cues, cannabinoid concentration, and duration of exposure. Furthermore, the functional properties of the differentiated MSCs need to be further investigated.

DRIVERS OF HETEROGENEITY IN PHYTOCANNABINOID-MEDIATED MSC PRIMING AND DIFFERENTIATION

Although multiple studies support the capacity of phytocannabinoids to modulate MSC behavior, the reported outcomes remain heterogeneous. This variability reflects not only biological differences between experimental models but also substantial methodological diversity across studies, which complicates direct comparison and interpretation of results.

One major source of heterogeneity arises from MSC origin and species. MSCs derived from distinct tissues (e.g., adipose tissue, bone marrow, periodontal ligament) and from different species (e.g., human, mouse, equine, canine) exhibit intrinsic differences in baseline immunophenotype, proliferative capacity, and differentiation potential. These characteristics can influence how MSCs respond to phytocannabinoid exposure, affecting both immunomodulatory priming and lineage-related transcriptional programs.

The baseline inflammatory context further contributes to divergent findings across studies. While some investigations deliberately establish an inflammatory microenvironment using stimuli such as LPS or pro-inflammatory cytokines (e.g., TNF-α) to assess the immunomodulatory capacity of phytocannabinoid-treated MSCs, others evaluate MSC responses under basal, non-inflammatory conditions. These experimental designs interrogate distinct biological states and are therefore not directly comparable.

Substantial heterogeneity is also introduced by differences in phytocannabinoid formulation and purity. While some investigations employ purified CBD or other isolated compounds, others utilize phytocannabinoid-rich extracts containing variable proportions of cannabinoids and secondary metabolites. Variations in plant chemotype, cultivation conditions, and extraction procedures can markedly alter phytochemical composition, potentially shifting the balance between priming-related immunomodulation and differentiation-related phenotypic changes.

Exposure parameters, including phytocannabinoid concentration and treatment duration, represent additional critical determinants of study outcomes. Available evidence consistently indicates a strong dose-dependent response, with higher phytocannabinoid concentrations more frequently associated with reduced MSC viability or overt cytotoxic effects. In contrast, lower concentrations are generally employed to investigate functional modulation, including immunoregulatory effects or differentiation changes. In the absence of standardized exposure protocols, variability in dose and duration alone may therefore lead to markedly different biological readouts, limiting cross-study comparability. Consequently, it is difficult to define an optimal concentration range for specific outcomes, highlighting the need for further standardized investigations.

Collectively, heterogeneity in MSC source, inflammatory context, phytocannabinoid formulation, exposure parameters, and endpoint selection provides a coherent explanation for the divergent findings reported across phytocannabinoid-based MSC priming and differentiation studies. Explicit recognition of these drivers is essential for accurate interpretation of existing data and for the rational design of future experimental protocols.

LIMITATIONS AND FUTURE PERSPECTIVES OF PHYTOCANNABINOIDS USE IN MSCS

There is increasing interest in employing isolated or semi-synthetic cannabinoids in cellular studies, primarily due to the challenges associated with obtaining standardized crude Cannabis sativa extracts and the need to attribute biological effects to specific compounds. Nonetheless, emerging evidence indicates that this approach should be interpreted with restraint. The proposed entourage effect, which posits that multiple cannabis-derived constituents (e.g., cannabinoids, terpenes) act synergistically, remains insufficiently supported by robust clinical or mechanistic data[61]. Therefore, further rigorous and well-controlled studies are required to determine whether full-spectrum preparations genuinely provide more consistent, enhanced therapeutic efficacy or improved safety profiles compared with isolated molecules.

However, the composition and concentration of phytocannabinoids can vary substantially according to regulatory policies, cultivation conditions, and extraction methods. For instance, some reports employ > 99% pure CBD, whereas others use cannabis extracts containing CBD along with low concentrations of THC. Such variability represents a critical confounding factor, as even small amounts of THC or other cannabis-derived constituents could modulate immunological responses, potentially explaining different findings attributed solely to CBD. Moreover, other secondary metabolites, such as terpenes and flavonoids, exhibit distinct pharmacological activities, supporting the potential of full-spectrum cannabis-based phytocomplexes. Major challenges ahead include selective breeding for targeted chemotypes and the standardization of growth and extraction procedures.

Despite their promising regulatory effects on MSCs, several limitations currently hinder the broader translational application of phytocannabinoids in regenerative medicine. Most available studies have focused almost exclusively on CBD, while few have examined THC or other cannabinoids such as CBG, CBC, and CBN as priming or differentiation agents. Evidence consistently indicates that MSC responses to phytocannabinoids are strongly dose-dependent, with lower concentrations generally promoting immunomodulatory or pro-differentiative effects, whereas higher concentrations are more frequently associated with reduced viability or cytotoxic responses. Interpretation of these outcomes is further complicated by substantial methodological heterogeneity across studies, including differences in phytocannabinoid purity (isolated compounds vs crude extracts), concentration ranges, treatment duration, and MSC origin (species and tissue source). This variability limits the comparability of similar biological phenotypes, such as immunomodulation or differentiation, and contributes to the fragmentation of the current literature. Collectively, these factors hinder the identification of optimal priming conditions and highlight the need for standardized experimental designs and harmonized priming protocols. Importantly, the pharmacological relevance of the concentration ranges commonly employed in vitro remains uncertain, as in many cases these doses have not been directly correlated with achievable tissue levels in vivo. Bridging this translational gap will require future investigations integrating pharmacokinetic and pharmacodynamic analyses to better align in vitro exposure paradigms with clinically relevant dosing scenarios.

From a mechanistic perspective, the molecular basis of phytocannabinoid-mediated MSC priming and differentiation remains largely undefined. In other cell systems, phytocannabinoids can signal through a broad repertoire of receptors, including canonical G protein-coupled receptors (CB1, CB2, GPR55, GPR119, GPR18), TRPV, and nuclear PPAR receptors, each associated with distinct downstream pathways and transcriptional programs[62]. However, whether these receptor systems operate similarly in MSCs, and how they connect to the signaling pathways that govern MSC plasticity, immunomodulation, and lineage specification, still requires further investigation.

Most current evidence on phytocannabinoid priming of MSCs derives from in vitro studies, which provide valuable mechanistic insights but have clear limitations regarding translational relevance. Only a few studies have explored in vivo models, which remain limited in scope and often do not evaluate the long-term safety, functional stability, or optimal administration routes of phytocannabinoid-primed MSCs. These limitations highlight the current boundaries of in vivo efficacy and safety and underscore the need for carefully designed investigations that assess not only differentiation outcomes but also the long-term functional integration, paracrine effects, and overall safety profile of primed MSCs. Future studies should consider clinically relevant delivery strategies, such as sustained-release systems, and standardized protocols to ensure reproducibility and reliability. Addressing these gaps will be essential to translate promising in vitro findings into robust regenerative medicine applications. Moreover, safety assessments in most studies remain largely restricted to short-term viability and proliferation assays. Comprehensive evaluation of additional safety endpoints, including persistent immunosuppressive effects, oxidative stress responses, and potential epigenetic alterations, will be critical to ensure the long-term functional stability and safety of phytocannabinoid-primed MSCs prior to clinical translation. Importantly, the long-term stability of cannabinoid-induced modifications in MSCs remains largely unexplored, including whether these effects persist after withdrawal of the stimulus or following transplantation. Future studies should incorporate experimental designs to monitor functional phenotypes, secretome profiles, and receptor expression over time.

Additionally, the inherent sensitivity of MSCs to environmental and pharmacological stimuli positions them as valuable models for studying cannabinoid-induced cytotoxicity, oxidative stress, and epigenetic alterations. Their multipotency and ease of isolation make them particularly suited to detect subtle molecular and functional changes that may not be evident in terminally differentiated cells, providing insight into cannabis-related cellular effects and potential long-term impacts on tissue homeostasis.

Future research should prioritize the establishment of standardized priming protocols, the comparative assessment of distinct phytocannabinoids and their combinations, and the definition of dose-response thresholds that ensure both efficacy and safety. In this context, increasing attention should be given to the MSC secretome, including EVs, as potentially safer and more controllable therapeutic products. To support translational development, future studies should incorporate standardized EV characterization, functional potency assays relevant to the intended therapeutic indication, and batch-to-batch quality control metrics suitable for release criteria under good manufacturing practice conditions.

CONCLUSION

Phytocannabinoids represent a promising strategy to enhance MSC therapeutic efficacy by modulating differentiation, survival, and immunoregulatory functions (Figure 3). Current evidence supports a modulatory, context-dependent effect, with CBD being the most extensively studied compound. Nonetheless, major knowledge gaps remain, including the mechanistic basis of receptor-mediated effects, long-term safety, and the roles of less-studied phytocannabinoids. Future research should prioritize standardized priming protocols, in vivo validation, and exploration of the MSC secretome and extracellular vesicles as safer alternatives to cell transplantation, paving the way for translational applications in regenerative medicine.

Figure 3 Graphical abstract. Created in BioRender. Amorim, R. (2026) https://BioRender.com/0lqgoi6. CBD: Cannabidiol; CBDA: Cannabidiolic acid; THC: Δ⁹-tetrahydrocannabinol; CBG: Cannabigerol; CBGA: Cannabigerolic acid; THCV: Tetrahydrocannabivarin; PPAR: Peroxisome proliferator activated receptor; CB1: Cannabinoid receptor type 1; CB2: Cannabinoid receptor type 2; MSCs: Mesenchymal stem cells.