Abstract
Background
Human mesenchymal stem cells from dental pulp (hMSC-DP), including dental pulp stem cells from permanent teeth and exfoliated deciduous teeth, possess unique MSC characteristics such as expression of specific surface molecules and a high proliferation rate. Since hMSC-DP have been applied in numerous clinical studies, it is necessary to establish criteria to evaluate their potency for cell-based therapies.
Methods
We compared stem cell properties of hMSC-DP at passages 5, 10 and 20 under serum (SE) and serum-free (SF) culture conditions. Cell morphology, proliferation capacity, chromosomal stability, surface phenotypic profiles, differentiation and immunoregulation ability were evaluated. In addition, we assessed surface molecule that regulates hMSC-DP proliferation and immunomodulation.
Results
hMSC-DP exhibited a decrease in proliferation rate and differentiation potential, as well as a reduced expression of CD146 when cultured under continuous passage conditions. SF culture conditions failed to alter surface marker expression, chromosome stability or proliferation rate when compared to SE culture. SF-cultured hMSC-DP were able to differentiate into osteogenic, adipogenic and neural cells, and displayed the capacity to regulate immune responses. Notably, the expression level of CD146 showed a positive correlation with proliferation, differentiation, and immunomodulation, suggesting that CD146 can serve as a surface molecule to evaluate the potency of hMSC-DP. Mechanistically, we found that CD146 regulates proliferation and immunomodulation of hMSC-DP through the ERK/p-ERK pathway.
Conclusion
This study indicates that SF-cultured hMSC-DP are appropriate for producing clinical-grade cells. CD146 is a functional surface molecule to assess the potency of hMSC-DP.
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Introduction
Mesenchymal stem cells (MSCs) have been used in clinics to treat a variety of human diseases [1,2,3,4]. MSCs can be isolated from multiple tissues, such as bone marrow, umbilical cord tissue, adipose tissue and dental pulp [5,6,7]. The minimal criteria for MSC identification were established by the International Society for Cellular Therapy (ISCT) in 2006 [8]. However, the standards for the quality assessment of MSCs from specific tissue resources have not yet been reported.
Human mesenchymal stem cells from dental pulp (hMSC-DP) have been isolated and extensively studied [9, 10]. Their superior proliferation, multi-differentiation, and immunomodulatory capacities have been reported [11,12,54]. In this study, we found that hMSC-DP at early passages such as P5 show optimal immunoregulation effects in vitro in a T cell coculture system and in vivo in a DSS-induced colitis mouse model. However, continued passaging (to P10 and beyond) reduces their therapeutic capacity for colitis mice, which may relate to their diminished ability to induce T cell apoptosis [28].
CD146 was initially identified as a specific marker of malignant melanoma [55]. Previous studies showed that CD146 is expressed on the surface of human bone marrow MSCs, human umbilical cord-derived MSCs, human adipose tissue-derived MSCs, DPSCs and SHED [36, 42, 56,57,58]. MSCs with high expression of CD146 show elevated osteogenic and immunoregulatory abilities compared to those with low CD146 expression [41, 42, 56]. Here, we found that the expression levels of traditional MSC surface markers, including CD73, CD90 and CD105, fail to reflect the potency of hMSC-DP, while the expression level of CD146 correlates with hMSC-DP capacity for proliferation, differentiation, and immunoregulation [26, 59]. Therefore, we propose CD146 as a functional surface molecule to predict the quality of hMSC-DP.
ERK/p-ERK pathway plays a critical role in regulating proliferation, immunoregulation and differentiation of MSCs [27, 60, 61]. Previous studies showed that CD146 expression is associated with the activation of ERK pathway during epithelial-mesenchymal transition [62] and tumor angiogenesis [63]. In this study, we demonstrated that CD146 maintained cell proliferation and immunomodulation through ERK/p-ERK pathway, but not osteogenic differentiation. Usually, a highly proliferative state is incompatible with differentiation in MSCs. The controversial role of ERK signaling has been discussed in the context of osteogenic differentiation of MSCs [45]. Activation of ERK signaling in human bone marrow-derived MSCs promotes osteogenic differentiation [64, 65], while upregulation of ERK/p-ERK pathway contributes to the suppression of osteogenesis of MSCs [27, 44, 46]. ERK/p-ERK may regulate other pathways, such as PI3-kinase/Akt or P38 pathway, to affect osteogenic differentiation of hMSC-DP [66, 67].
The minimal criteria for defining MSCs proposed by ISCT are quite basic and general [8], but it may fail to totally reflect the comprehensive characteristics of MSCs, such as their trophic activity [68] and immunomodulatory capacity [28]. Therefore, it may be insufficient to predict the potency of MSCs for clinical applications. Moreover, different tissue-derived MSCs may exhibit parent tissue specificity and possess different functional potential [69]. With the improvement of our understanding of the functional and tissue-specific characteristics of MSCs, it is necessary to define the criteria of tissue-specific MSCs for translational precision therapies. To meet upcoming requirements for defining optimal MSCs for clinical application, we propose additional criteria to define the potency of hMSC-DP: (1) Adherence to plastic forming CFU-F in serum-free culture conditions; (2) CD146 expressed by over 30% of cells; (3) Neural differentiation potential. These added criteria aim for standardized identification of hMSC-DP for clinical use. In our study, we found that 10–80% of SHED and 5–70% of DPSCs expressed CD146, which was positively correlated with stem cell function. Some critical capacities of hMSC-DP were significantly decreased with the reduced expression of CD146 at passage 10. The positive rate of CD146 expression detected by flow cytometry was about 30–40% at passage 10 in our study. Therefore, we propose that CD146 can serve as a functional surface molecule to evaluate the potency of hMSC-DP. When CD146 positive rate is above 30%, hMSC-DP can provide optimal therapeutic effect in DDS-induced colitis mouse model.
Conclusion
We explored the physiological and functional status of hMSC-DP in the SF culture system and established minimal criteria to identify the potency of hMSC-DP for potential clinical application. CD146 is a functional surface molecule that reflects the potency of hMSC-DP.
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its Additional files.
Abbreviations
- hMSC-DP:
-
Human mesenchymal stem cells from dental pulp
- DPSCs:
-
Postnatal human dental pulp stem cells
- SHED:
-
Stem cells from human exfoliated deciduous teeth
- SE:
-
Serum
- SF:
-
Serum-free
- MSCs:
-
Mesenchymal stem cells
- ISCT:
-
International Society for Cellular Therapy
- SOPs:
-
Standard operation procedures
- FBS:
-
Fetal bovine serum
- EdU:
-
5-Ethynyl-20-deoxyuridine
- PD:
-
Population doublings
- CFU:
-
Colony-forming unit
- WGA:
-
Wheat germ agglutinin
- β-gal:
-
β-Galactosidase
- SA-β-gal:
-
Senescence-associated β-galactosidase
- Runx2:
-
Runt-related transcription factor 2
- ALP:
-
Alkaline phosphatase
- PPARγ:
-
Peroxisome proliferator-activated receptor-γ2
- LPL:
-
Lipoprotein lipase
- DSS:
-
Dextran Sulfate Sodium
- DAI:
-
Disease activity index
- HAI:
-
Histological activity index
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Acknowledgements
We thank Mr. Lulu (South China Center of Craniofacial Stem Cell Research, Sun Yat-Sen University) for his kind help in this study.
Funding
This study was supported by grants from the National Natural Science Foundation of China (81700928 to L.M.), the Youth Teacher Training Project of Sun Yat-sen University (17ykpy71 to L.M.), the Pearl River Talent Recruitment Program (2019ZT08Y485 and 2019QN01Y138 to S.S., 2019JC01Y182 to X.K.), the Guangdong Financial Fund for High-Caliber Hospital Construction (174-2018-XMZC-0001-03-0125, D-07 to S.S., D-11 to X.K.), the National Science and Technology Major Project of the Ministry of Science and Technology of China (2018ZX10302207), the Sun Yat-sen University Young Teacher Key Cultivation Project (18ykzd05 to X.K.) and the Natural Science Foundation of Guangdong (2016A030313262 to X.M.).
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LM and ZH contributed to designing the study plan, performing experimental procedures and drafting the manuscript. DW contributed to data acquisition, analysis and interpretation. XK and XM contributed to data analysis and interpretation. SS contributed to the project conception, experimental design, writing manuscript and supervision. All authors approved the final version of the manuscript.
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Human samples collection was approved by the Medical Ethics Committee of Hospital of Stomatology, Sun Yat-sen University (Protocol Number: KQEC-2020-055-01). All animal experiments were performed under an institutionally approved protocol for the use of animal research of Sun Yat-sen University (Protocol Number: SYSU-IACUC-2020-000394).
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Supplementary Information
Additional file 1: Fig. S1
Characterization of the morphology of hMSC-DP under SF culture conditions. a-b Cell morphology analysis by high-content imaging. The nuclear area (a) and cell area (b) of DPSCs and SHED are shown in SE and SF culture conditions. n = 3 for each group. c Cellular senescence assay. The percentage of SA-β-gal positive cells was calculated and compared between SE and SF culture conditions. n = 3 ~ 5 for each group. SE, serum; SF, serum-free. Data shown as mean ± SEM. ns, not significant. Fig. S2 Proliferation capacity of hMSC-DP under SF culture conditions. a CFU-F assay. The numbers of colonies of hMSC-DP under SE or SF culture conditions were calculated and compared. n = 3 for each group. b Population doubling scores were calculated and compared between SE and SF culture conditions in DPSCs and SHED groups, independently. n = 3 for each group. c EdU assay. The percentage of EdU-positive cells were calculated and compared between SE and SF culture conditions in DPSCs and SHED groups, independently. n = 3 for each group. Scale bar = 200 μm. SE, serum; SF, serum-free. Data shown as mean ± SEM. *p < 0.05. ns, not significant. Fig. S3 Surface phenotypic profiles and in vitro immunoregulation ability of hMSC-DP. a-b Flow cytometry showed the percentage of CD146-positive hMSC-DP under SE or SF culture conditions at P5, P10 and P20 (a). The percentage of CD146-positive cells was compared between SE and SF culture conditions (b). n = 3 for each group. c The percentage of apoptotic T cells was calculated in SE and SF culture conditions. n = 3 for each group. SE, serum; SF, serum-free. Data shown as mean ± SEM. *p < 0.05. ns, not significant. Fig. S4 Multilineage differentiation of hMSC-DP. a Alizarin red staining assay. The osteogenic capacity was compared between SE and SF culture conditions at P5, P10 and P20. n = 3 for each group. b Oil red O staining assay. The adipogenic capacity of hMSC-DP was compared between SE and SF culture conditions at P5, P10 and P20. n = 3 for each group. Scale bar = 50 μm. SE, serum; SF, serum-free. Data shown as mean ± SEM. **p < 0.01. ns, not significant. Fig. S5 Therapeutic effects of hMSC-DP on experimental colitis. a Disease activity index (DAI) of DPSC- and SHED-treated groups was compared between SE and SF culture conditions at P5, P10 and P20. n = 3 for each group. b The colon length after DPSCs and SHED treatment was compared between SE and SF culture conditions at P5, P10 and P20. n = 3 for each group. c Histological structure was examined by H&E staining. The histological score of each group was compared between SE and SF culture conditions. n = 3 for each group. Scale bar = 100 μm. SE, serum; SF, serum-free. Data shown as mean ± SEM. ns, not significant. Fig. S6 The expression level of CD146 is associated with hMSC-DP properties. a The correlation of CD146 with experimental parameters. b Linear regression plots showed the correlation between CD146 expression and experimental parameters including CFU-F clones, percentage of EdU positive cells, percentage of Alizarin red positive area, percentage of SA-β-gal positive cells, DAI scores, HAI scores and colon length.
Additional file 2
. Table S1. Summary of clinical trials of hMSC-DP.
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Ma, L., Huang, Z., Wu, D. et al. CD146 controls the quality of clinical grade mesenchymal stem cells from human dental pulp. Stem Cell Res Ther 12, 488 (2021). https://doi.org/10.1186/s13287-021-02559-4
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DOI: https://doi.org/10.1186/s13287-021-02559-4