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

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.

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.