Research ArticleExperimental Studies
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Improved Cell Properties of Human Dental Pulp Stem Cells (hDPSCs) Isolated and Expanded in a GMP Compliant and Xenogeneic Serum-free Medium

JUAN LI, XUEWEI LV, TINGTING GE, JIAMAN SHI, GIDEON VERWOERD, HAIYAN LIN and YUANSONG YU
In Vivo November 2023, 37 (6) 2564-2576; DOI: https://doi.org/10.21873/invivo.13364

Abstract

Background/Aim: Human dental pulp mesenchymal stem cells (hDPSCs) are considered to be a good cell source for cell-based clinical therapy, due to the advantages of high proliferation capacity, multilineage differentiation potential, immune regulation abilities, less ethnic concerns and non-invasive access. However, hDPSCs were traditionally isolated and expanded in medium containing fetal bovine serum (FBS), which is a barrier for clinical application due to the safety issues (virus transmission and allergy). Although many studies make efforts to screen out a suitable culture medium, the results are not promising so far. Therefore, a standard good manufacturing practice (GMP) compliant culture system is urgently required for the large-scale cell production. This study aimed to find suitable culture conditions for producing clinical grade hDPSCs to meet the requirements for clinical cell-based therapy and further to promote the application of hDPSCs into tissue regeneration or disease cure. Materials and Methods: We derived hDPSCs from nine orthodontic teeth expanded in two different media: a GMP compliant and xenogeneic serum-free medium (AMMS) and a serum containing medium (SCM). Cell propterties including morphology, proliferation, marker expression, differentiation, stemness, senescence and cytokine secretion between these two media were systematically compared. Results: hDPSCs cultured in both media exhibited the typical characteristics of mesenchymal stem cells (MSCs). However, we found that more cell colonies formed in the primary culture in AMMS, and the hDPSCs displayed higher proliferation capacity, differentiation potential and better stemness maintenance during sub-culturing in AMMS. Conclusion: Cell properties of hDPSCs could be improved when they were isolated and expanded in AMMS, which might provide a good candidate of culture medium for large-scale cell manufacturing.

Key Words:

Since mesenchymal stem cells (MSCs) were isolated from bone marrow for the first time in 1968 (1), divergent MSCs have been identified from various tissues and the minimal criteria defining MSCs have been recommended by the International Society for Cellular Therapy (ISCT) which include plastic adhesive growing, cell surface marker expression and mesodermal differentiation (2). Due to their regenerative properties, MSCs have attracted considerable attention in the field of clinical medicine, and extensive studies have been performed to investigate their safety and efficacy (3). According to the US National Institute of Health–Clinical Trials database (http://clinicaltrials.gov) (3) between 2015 to 2021, 416 clinical trials based on the use of MSCs have been implemented to investigate treatment of various refractory diseases, therefore an accumulating body of evidence suggests a promising future for MSCs based therapy.

Human dental pulp stem cells (hDPSCs) are a population of cells isolated from adult permanent teeth, which strictly complies with MSCs characteristics (4). The high proliferation and self-renewal ability, multi-lineage differentiation potential, cytokine secretion and immunomodulation of these cells have been well studied (5-6). Apart from the typical mesodermal tri-lineage differential potential, hDPSCs could also differentiate into neural cells (4, 7), vascular endothelial cells (8), pancreatic islet cells (9) and liver tissue cells (10). Particularly since hDPSCs originate from neural ridge cells, the high differentiation potential into neural cells may indicate a better effectiveness in nerve damage repair (11). In addition, hDPSCs have a potent immunomodulatory ability, and the transplanted cells effectively improve the inflammatory environment of organisms through paracrine or cell contact mechanisms. Based on their differentiation potential and immunomodulatory ability, clinical trials on a variety of diseases, including neurodegenerative diseases, diabetes and immune disorders have been started (12-13).

Although the therapeutic significance of hDPSCs and other MSCs has been demonstrated, a standard operation procedure (SOP) complying with good manufacturing practice (GMP) is now urgently required (14). Since the culture medium is closely related to cell viability, properties, and functions, an optimized and GMP compliant culture medium is a key factor for large scale cell production. Conventionally, MSCs have been cultured in media supplemented with fetal bovine serum (FBS) to support cell survival and expansion. However, animal or allogeneic serum may cause allergic problems (15), and risks of animal derived pathogen transmission during cell-based therapy (16).

In order to meet the requirements of clinical application, studies have tried to culture hDPSCs in a 3-D spheroids system with chemical fully defined medium, which is believed to be better for stemness maintenance, proliferation capacity and the differential potential of the cells (5, 8, 17). However, in these studies the cells were isolated in medium supplemented with either FBS (17) or cytokines (5, 8) to keep the cells at a specific differential orientation. Efforts have also been devoted to screen out a suitable commercially available, GMP compliant and xenogeneic serum-free medium, which may bolster large scale cell manufacturing (16, 18). Currently results are not ideal, because either the proliferation and colony forming unit (CFU) counts have been reduced (18) or the immunotype has not been strictly matched with the definition of MSCs (16). AMMS®MSC Cell Culture Kit 2.0 (AS-13, AMMS, T&L biotechnology, Beijing, PR China) is a commercially available GMP compliant and xenogeneic serum-free medium, including basic medium (AD09-2) and manufacture provided supplements (AS13-1). Here, we isolated and sub-cultured hDPSCs in FBS containing medium (SCM) or AMMS medium in parallel, and comprehensively compared proliferation capacity, immuno-phenotypes, differentiation potential, stemness maintenance and cytokine secretion of the cells expanded under these two different culture conditions.

Materials and Methods

Isolation and expansion of hDPSCs. Orthodontic teeth were collected from donors aged 19 to 30 years old at the orthodontic Department of the Hangzhou Stomatology Hospital. All the donors had been fully informed about the usage of teeth and a consent form was signed by each donor. The hDPSCs were isolated and cultured following a protocol adapted from a previous report (4). Briefly, the teeth without severe damage or caries were physically split and the complete pulp tissues were extracted with broaches. After it had been minced with blades, the pulp tissues were digested with 3 mg/ml collagenase type I (Gibco, Grand Island, NY, USA) and 4 mg/ml dispase type II (Gibco) (collagenase I: dispersive enzyme=1:1) for 1 h at 37°C. The digestions were terminated with α-MEM and the suspensions were evenly divided between two tubes, then pelleted down by centrifugation at 300×g. One tube of cells was suspended in α-MEM media (Hyclone, Thermo Fisher Scientific Inc, Waltham, MA, USA) supplemented with 10% FBS (Gibco),100 mM ascorbic acid 2-phosphate, 2 mM GlutaMax (Gibco), 50 U/ml gentamicin (serum containing medium, SCM). The other tube of cells was suspended in AMMS basic medium plus manufacture provided supplements (AMMS, T&L biotechnology). Cells in both media were seeded in 25 cm2 flasks (Nunc, Thermo Fisher Scientific Inc) and maintained under standard conditions (37°C, 100% humidity, 5% CO2). Cell colonies were counted on the sixth day of the primary culture (only colonies with 5 or more cells were included). The media were changed every 3 days, and all the cells were passaged around 10 days of primary culture. During sub-culturing, the cells were counted with a hemocytometer (Thermo Fisher Scientific Inc) and replated every passage through to P5. Population doubling time (PDT) was determined at each passage using the following formula:

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Colony forming unit (CFU) assay. CFU assays were performed on hDPSCs at P2 and P5. Briefly, hDPSCs were seeded into a 6-well plate (Nunc) at a density of 100 cells/well and cultured in SCM or AMMS for 2 weeks. Then they were fixed and labeled with crystal violet (Solarbio, Beijing, PR China) for 3-5 min at room temperature, and colonies (over 50 cells) were counted. The CFU rates were determined as CFU rate=(number of colonies/number of cells inoculated)×100%.

Immuno-phenotypic analysis by flow cytometry. hDPSCs at P2 and P5 were collected and washed with PBS (Solarbio), and the cells were labeled with fluorescein isothiocyanate-conjugated antibodies against human CD73, CD90, CD146, CD105, CD45 (Biolegend Inc, San Diego, CA, USA), CD34, CD14, CD19 and HLA-DR (BD Biosciences, Qume Drive, San Jose, CA, USA) for 30 min at room temperature. Flow-cytometric analyses were performed with the NovoCyte Quanteon system (Agilent, Beijing, PR China) and NovoExpress software (version 1.6.1).

Tri-lineage differentiation analyses. For osteogenic and adipogenic differentiation analyses, hDPSCs at P5 were seeded into a 12-well plate (Nunc) at a density of 1.5×104 cells/well and cultured in SCM and AMMS. After having reached 80% confluence, the cells were cultured in the osteogenic induction solution (OriCell® Human Umbilical Cord Mesenchymal Stem Cell Osteogenic Induction and Differentiation Kit, Cyagen, Guangzhou, PR China) or the adipogenic induction solution (OriCell® Human Umbilical Cord Mesenchymal Stem Cell Adipogenic Induction and Differentiation Kit, Cyagen). As a control, the cells were cultured in SCM or AMMS lacking the osteogenic or adipogenic supplements. After 3 weeks of induction, the calcium depositions were stained with alizarin red (Cyagen), and the lipid droplets were stained with Oil Red O (Cyagen). For chondrogenic differentiation analysis, hDPSCs at P5 were precipitated by centrifugation (500×g for 6 min), and the precipitations were cultured in the chondrogenic induction solution (OriCell® Human Umbilical Cord Mesenchymal Stem Cell Chondrogenic Induction and Differentiation Kit, Cyagen) in a 15 ml centrifuge tube (Nunc) (18). As a control, the precipitation of cells was cultured in SCM or AMMS lacking the chondrogenic supplements. After 3 weeks, the formed pellets were buried with OCT (Sakura Finetek USA Inc, Torrance, CA, USA) and sliced with a frozen microtome (Thermo Fisher Scientific Inc). The sections were stained with alicin blue (Cyagen).

Neural cell oriented differentiation assay. hDPSCs at P5 were seeded into 24-well plates (Nunc) coated with 0.1% gelatin (Solarbio) at 1.5x104 cells/well and cultured in SCM and AMMS. When they reached 80% confluence, the cells were cultured in Neurobasal medium (Gibco) supplemented with 1X B27 (Gibco), 20 ng/ml EGF (Genever, Taizhou, Jiangsu, PR China), 40 ng/ml 2565 control, cells continued to be cultured in SCM or AMMS. After 2 weeks, the cells were fixed and blocked in the PBS with 5% normal goat serum (Sigma-Aldrich, Burlington, MA, USA) and 0.1% Tritonx-100 (Sigma-Aldrich) for 1 h. Then they were incubated with the following primary antibodies diluted in PBS with 1.5% goat serum and 0.1% Tritonx-100 at 4°C overnight: Anti-Nestin Antibody (Boster, Wuhan, Hubei, PR China, 1:200), Anti-vimentin Antibody (Boster, 1:200), Anti-GFAP Antibody (Abcam, Shanghai, PR China, 1:200) and Anti-NeuN Antibody (Abcam, 1:200). After being rinsed with PBS, they were incubated with secondary antibodies for 1 h at room temperature: DyLight 488 Conjugated AffiniPure Donkey Anti-mouse IgG (H+L) (Boster, 1:500), DyLight 550 Conjugated AffiniPure Goat Anti-mouse IgG (H+L) (Boster, 1:500). The plates were sealed with Antifade Mounting Medium (Beyotime, Shanghai, PR China).

The mounted plates were observed and taken photos under the fluorescence inverted microscope (OLYMPUS IX73, Olympus Corporation, Tokyo, Japan). In order to analyze the fluorescence intensity per cell and ratio of positive cells, the parameters (excitation, emission, fluorescence intensity, exposure time) were kept exactly the same for all experiments when the photos were taken, and the analyses strictly followed the principles suggested by Ellen C Jensen (19).

Enzyme-linked immunosorbent assay. hDPSCs at P5 were seeded into 25 cm2 flasks with SCM or AMMS. When the cells reached 80% confluence, both media were replaced with α-MEM and culture continued for another 48 h. Conditioned media were collected to analyze the levels of human HGF, FGF2, BDNF, GDNF, IL-6, IL-10, TGFβ1 and PGE2 with the ELISA kits (Boster; Nanjing Jiancheng, Nanjing, PR China) according to the manufacturer’s protocol. Each analysis was analyzed in duplicate, and the absorbance was measured at 450 nm on a Microplate Reader (Thermo Fisher Scientific Inc).

Real-time polymerase chain reaction (Real-time PCR). The total RNA was isolated (RNAprep Pure Cell/Bacteria Kit, TIANGEN, Beijing, PR China) and quantified spectrophotometrically (UV5Nano, MettlerToledo, Greifensee, Switzerland). The cDNA synthesis was conducted according to PrimeScript™ RT Master Mix kit (Takara Bio Inc, Beijing, PR China). Subsequently, real-time PCR assay was performed using TB Green® Premix Ex Taq™ II kit (Takara) on a 7500 Real-Time PCR Systems (Applied Biosystems, Thermo Fisher Scientific Inc) according to the manufacturer’s protocols. Data were then processed using the 2−ΔΔCT method (20). The expression of each target gene was normalized to that of the RPS18 housekeeping gene and expressed as fold change relative to the control group. All the primer pairs (Tsingke Biotech Co. Ltd, Beijing, PR China) employed in this study are listed in Table I.

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Table I.

The primer sequences for real-time PCR.

SA-β-gal assay. The senescence of hDPSCs cultured in SCM and AMMS were analyzed by the cell SA-β-gal staining kits (Beyotime) following the manufacturer’s instructions. In brief, cells at P5 were seeded in a 6-well plate at 5×104 cells/well with the corresponding medium. Cells were fixed with paraformaldehyde (Solarbio) at 80% confluence for 15 min and then incubated overnight with working solution (10 μl staining solution A, 10 μl staining solution B, 930 μl staining solution C, 50 μl X-Gal solution) at 37°C without CO2. The senescent cells were identified as blue-stained cells by microscopy and the positive cell rate was calculated.

Statistical analyses. The values of all experiments were expressed as the mean±standard error (SE). Student’s t-test was used to analyze the results with IBM SPSS statistical software (version 22.0), and all figures were taken by GraphPad Prism 9. p<0.05 indicated a statistically significant difference.

Results

hDPSCs isolation in SCM and AMMS. In this study, we divided the digested dental pulp tissues in two parts and implanted each part into normal culture dishes with SCM and AMMS medium. The individual cell colonies were observed at D6 (Figure 1) during primary culture. Detailed information about the teeth and the donors are listed in Table II. The results showed that primary colonies formed from all the samples in both media (Figure 1), but the statistical analysis showed that the number of colonies formed in AMMS was significantly higher than that in SCM (Table II, Figure 1 a2; SCM vs. AMMS, 18.33±15.85 vs. 25.22±15.95, p=0.003). Although the age of the donors may affect cell properties (21), the statistical analysis did not show a significant relationship between cell colony formation and the age of the donors, which might be due to the limited sample size or the strict selection of the age range (data not shown). Primary colonies formed in all the samples, but some cell lines failed to expand to P5 with normal morphology due to differentiation, and the successful isolation rates were not significantly different between the cells cultured in SCM and AMMS (SCM vs AMMS: 5/9 vs. 8/9, p=0.294) (Table II, Figure 1 a3). However, after analyzing the data, it seems likely that a larger sample size would show a higher isolation success rate in AMMS.

Figure 1.
Figure 1.

Isolation and expansion of hDPSCs in SCM and AMMS. (a1) Primary colony morphology observed at day 6 during P0 culture; (a2) Comparison of primary colonies formed at day 6 (p=0.003); (a3) Comparison of isolation success rate (p=0.294); (b1) Cells sub-cultured in SCM and AMMS; (b2) Population doubling times of cells passaged from P1 to P5; (c1) Colonies stained with crystal violet; (c2) Percentage of CFU formation of cells at P2 and P5 (all p>0.05) (Scale bar=200 μm). hDPSCs: Human dental pulp stem cells; SCM: serum containing medium; AMMS: serum/xeno-free medium (T&L biotechnology, Beijing, PR China); CFU: colony forming unit.

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Table II.

Human dental pulp mesenchymal stem cells (hDPSCs) isolation results and their relationship with the teeth resources.

Proliferation capacity of hDPSCs isolated and expanded in SCM and AMMS medium. The hDPSCs sub-cultured in both media adhered to the bottom of the dish and displayed a classic spindle shape morphology, but the cells in AMMS seem slenderer (Figure 1 b1), which has also been noticed in another study of hDPSCs cultured in a xenogeneic serum-free medium (16). The population doubling times (PDT) were analyzed on the hDPSCs expanded from P1 to P5. At P1, the PDT of cells cultured in SCM and AMMS was 28.11±4.50 h and 28.55±3.10 h respectively (p=0.625). With passaging, the PDT of cells in both media gradually increased, and the PDT of cells cultured in SCM increased more rapidly. By the time of P5, the PDT of cells in SCM was significantly higher (Figure 1 b2; SCM vs. AMMS: 112.58±36.8 h vs. 57.62±3.92 h, p=0.001).

CFU formation is a good indicator of the proliferation or self-renewal capacity of a cell line. In this study, we counted the number of cell colonies formed by the hDPSCs at P2 and P5. The analyses showed that there were no significant differences of the CFU formation rates between the hDPSCs cultured in SCM and AMMS, as well as between the hDPSCs at P2 and P5 cultured in the same medium (Figure 1 c2). However, distinctive colony morphologies were observed. The colonies formed by the cells cultured in SCM were more stereoscopic and aggregated, while colonies formed by the cells cultured in AMMS were flat, loose, and larger. By the time they were dying, some colonies had already started to merge with each other (Figure 1 c1), which may indicate a higher proliferation capacity of the cells cultured in AMMS.

Immuno-phenotype of hDPSCs. According to the definition of MSCs proposed by ISCT, we tested the immunophenotypes of hDPSCs at P2 and P5 by flow cytometry, including the surface markers of CD73, CD90, CD105, CD45, CD14, CD19, CD34, HLA-DR. The results showed that hDPSCs expanded in both media strongly expressed CD73, CD90 and CD105 (Figure 2, >95%) and barely expressed CD45, CD14, CD19, CD34 and HLA-DR (Figure 2, <2%). Statistical analyses showed that there was no significant difference of expression percentage between the cells cultured in SCM and AMMS (all p>0.05), as well as no difference between the cells at P2 and P5 cultured in the same medium (Figure 2). In addition, CD146 is related to the proliferation and differentiation ability of stem cells in vitro and in vivo (22, 23). In this study we also detected CD146 expression in the cells and the results showed that CD146 expression positive rates were as high as 85%, which may indicate a decent capacity of proliferation and differentiation of the cells cultured in our system (22, 23).

Figure 2.
Figure 2.

Expression of immunophenotypic markers of hDPSCs cultured in SCM and AMMS. Negative control (green). hDPSCs: Human dental pulp stem cells; SCM: serum containing medium; AMMS: serum/xeno-free medium (T&L biotechnology, Beijing, PR China).

Stemness and senescence analyses. Stemness maintenance is a good feature to evaluate the cell quality and suitability of the cell manufacturing process. Here we detected by real time-PCR the expression of the pluripotential marker genes NANOG, OCT4 and SOX2. Neither the cells at P2 nor at P5 showed any differences in expression of any of these three genes between cells cultured in SCM and AMMS. However, the sub-culturing to P5 did cause a significant decrease of SOX2 expression of the cells cultured in SCM (p=0.002), but not for the cells cultured in AMMS (Figure 3a). These results may reflect a slightly better stemness maintenance when cells were cultured in AMMS.

Figure 3.
Figure 3.

Analyses of stemness associated genes (NANOG, OCT4, SOX2) expression and cell senescence during sub-culturing. (a) The relative mRNA expression comparison between cells cultured in SCM and AMMS at P2 and P5 (the expression of different genes cultured in SCM at P2 served as control); (b) SA-β-GAL staining (cells with blue color indicating the senescence); (c) The comparison of the ratio of SA-β-gal positive cells to total cells when cultured in SCM versus AMMS at P5 (p=0.97) (Scale bar=200 μm). SCM: Serum containing medium; AMMS: serum/xeno-free medium (T&L biotechnology, Beijing, PR China).

The cell senescence during expansion was detected by dying based on the SA-β-gal activity (Figure 3b). The detection showed that the rates of senescent cells were quite low (SCM: 9.92±2.54%; AMMS: 10.03±2.81%) in the cells cultured to P5 both in SCM and AMMS, which is comparable to other studies (14) (Figure 3c).

Cytokine secretion. Enzyme linked immunosorbent assay (ELISA) was used to detect the cytokine secretion in hDPSCs at P5, including HGF, FGF2, BDNF, GDNF, IL-6, IL-10, PGE2 and TGFβ1. The results showed that the expression of HGF of the cells cultured in AMMS was higher, while the expression of PGE2 and TGFβ1 was lower than the cells cultured in SCM (Table III). This difference in cytokine secretion of the cells produced in different culture conditions may affect their effectiveness in cell-based therapy.

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Table III.

Cytokine expression of human dental pulp mesenchymal stem cells (hDPSCs) cultured in serum containing medium (SCM) and serum/xeno-free medium (AMMS).

Tri-lineage differentiation potential in vitro. Alizarin red staining clearly demonstrated a large amount of calcium deposits in hDPSCs both cultured in SCM and AMMS after induction (Figure 4a), which reflects a high osteogenic differentiation capacity. Furthermore, we detected and analyzed the relative expression of osteogenic genes of ALP and RUNX by real time-PCR. The results showed that the expression of both genes in cells cultured in AMMS is higher than that in SCM without osteogenic induction; and both cells responded to the induction significantly increasing the expression of ALP. However, ALP expression in the cells cultured in AMMS after the induction was significantly higher than that in SCM (Figure 4b).

Figure 4.
Figure 4.

The tri-lineage differentiation potential of hDPSCs cultured in SCM and AMMS at P5. (a) The osteogenic, adipogenic, and chondrogenic capacities of hDPSCs were shown with alizarin red, oil red O and alicin blue staining respectively. (b) The relative mRNA expression level of osteogenic related gene (ALP, RUNX2); (c) The relative mRNA expression level of adipogenic related gene (LPL, PPARγ); (d) The relative mRNA expression level of chondrogenic related gene (ACAN, SOX9) (Scale bar=200 μm, *p<0.05). hDPSC: Human dental pulp stem cells; SCM: serum containing medium; AMMS: serum/xeno-free medium (T&L biotechnology, Beijing, PR China).

The Oil red O staining showed a very limited adipogenic capacity in hDPSCs cultured in SCM after the induction, but the cells cultured in AMMS clearly exhibited more lipid droplets responding to the induction (Figure 4a), which indicates a higher adipogenic capacity of hDPSCs cultured in AMMS. Consistently, the relative expression of adipogenic genes of LPL and PPARγ in the cells cultured in AMMS after induction was significantly higher than that in the cells cultured in SCM, although the expression of both genes increased responding to the induction (Figure 4c).

With or without cartilage induction, chondrocyte spheres were formed by hDPSCs cultured in both media, but alician blue staining showed that the spheres formed by hDPSCs cultured in AMMS without induction were smaller and less condensed (Figure 4a). This phenomenon reflects that the hDPSCs expanded either in SCM or in AMMS in this study could spontaneously differentiate into chondrocytes, but this spontaneous differentiation might be less in hDPSCs cultured in AMMS. Consistently, the cells cultured in SCM without induction did show a higher ACAN expression. Both genes’ expression increased significantly in the cells cultured in SCM responding to the induction, but in AMMS culture this response was only found in ACAN expression (Figure 4d).

Neural cell oriented differentiation. Consistent with previous studies (7, 24), hDPSCs cultured in SCM could spontaneously differentiate into neural lineage cells, and the immuno-staining showed amounts of cells expressing Nestin (neuronal progenitor marker), Vimentin (glial progenitor marker) and NeuN (mature neuronal marker), but not GFAP (mature glial marker). Surprisingly, hDPSCs cultured in AMMS only demonstrated Nestin and Vimentin staining (Figure 5a), and both the ratio of positive cells and fluorescence intensity per cell of Nestin were significantly higher than the hDPSCs cultured in SCM. This exclusive expression of immature neural cell markers can also be induced by EGF and bFGF treatment on the cells cultured in SCM. But after neural induction with EGF and bFGF, the fluorescence intensity per cell of both Nestin and Vimentin staining in the cells cultured in AMMS was still higher than that in SCM, which indicates a higher expression level of Nestin and Vimentin in the cells cultured in AMMS after induction (Figure 5b,c). The higher level of expression of Nestin and Vimentin could be a sign of better stemness maintenance in AMMS culture.

Figure 5.
Figure 5.

Neural oriented differentiation potential of hDPSCs cultured in SCM and AMMS. (a) The neural stem/progenitor cell markers (Nestin, Vimentin) and mature neural cell markers (NeuN, GFAP) were stained (Scale bar=100 μm); (b) Rates of Nestin and Vimentin positive expressing cells cultured in SCM and AMMS (*p<0.05); (c) Single cell fluorescence intensity of expression of Nestin and Vimentin (*p<0.05). hDPSCs: Human dental pulp stem cells; SCM: serum containing medium; AMMS: serum/xeno-free medium (T&L biotechnology, Beijing, PR China).

Discussion

Traditionally hDPSCs have been cultured in media supplemented with FBS. However, this is not encouraged to be used in cell manufacturing due to the risk of immunoreaction and virus infections (15-16). Hence, a standard and GMP compliant process with a commercially available xenogeneic serum-free medium for MSCs production is currently required in order to meet the need for clinical cell-based therapy (16). For this purpose, we isolated and expanded hDPSCs in a serum containing medium (SCM) and a commercially available, GMP compliant, xenogeneic serum-free medium (AMMS) in parallel, and comprehensively compared the properties of the cells cultured in these media.

In order to reduce the influence of variations between different cell batches, the digested pulp tissues were divided equally into two groups, then cultured in SCM and AMMS in parallel. The number of cell colonies formed during primary culture was comparable to the previous study (4), but more primary colonies formed in AMMS than in SCM, which may indicate a better micro-environment for colony formation in AMMS. Although a previous study showed that the age of the donors may affect the success rate of isolation (21), we did not find a statistically significant correlation between them, probably due to the limited sample size and the strict age selection. Probably for the same reason there was no significant difference of isolation success rates between SCM and AMMS, although the intuitive data showed higher success rates in AMMS. Understandably the divided culture caused a clear reduction in the primary colony number compared with isolation from the whole pulp tissue (data not shown). This means that the primary culture would take longer period and need more division times (probably double) to reach the suitable confluence. For this reason, the sub-culturing to P5 in this study may be comparable to P10 for normal hDPSCs isolation and expansion.

Similar to other studies (14, 16), the PDT of hDPSCs gradually increased during sub-culturing in both SCM and AMMS, but the PDT for cells cultured in SCM increased faster. By the time of P5, the PDT of hDPSCs cultured in SCM was significantly longer. Although cells cultured in both media had a very good ability to form colony units, the size of the colony was clearly larger for the cells cultured in AMMS. This distinctive morphology may be caused by high levels of HGF (25), which is consistent with the higher secretory level of HGF in AMMS. These data demonstrated that the hDPSCs isolated in AMMS displayed a higher proliferation and self-renewal capacity compared to the hDPSCs isolated in SCM.

The surface marker expression of hDPSCs isolated in this study strictly complied with the definition of MSCs recommended by ISCT (2). However, it is interesting that the cells displayed a very high percentage of CD146 expression compared to other reports (22, 23). It has been reported that the expression level of CD146 in hDPSCs is positively correlated with proliferation, differentiation, and immunomodulation (22, 23), and CD146 positive cells have higher osteogenic differentiation capacity (22). The high percentage of CD146 positive expression may reflect the good cell properties of hDPSCs obtained following the isolation and expansion process in this study.

Culture conditions are critical for maintaining the stemness properties of the cells during expansion, which might have an impact on the MSC clinical performance (14, 18). Transcription factors NANOG, OCT4, SOX2, and NESTIN are expressed in pluripotent cells and they play a crucial role in maintaining characteristics of stemness such as self-renewal and pluripotency (26). The real-time PCR data showed that hDPSCs sub-cultured to P5 in AMMS could maintain the expression of OCT4, NANOG and SOX2, but the expression of SOX2 in hDPSCs sub-cultured to P5 in SCM reduced significantly compared to P2 hDPSCs sub-cultured in SCM. Especially, the immunostaining results clearly showed a higher level and percentage of positive cells of NESTIN expression in hDPSCs sub-cultured in AMMS than those sub-cultured in SCM. These results support the idea that AMMS may maintain the stemness properties of hDPSCs better during expansion.

Many studies demonstrated the differentiation capacity of hDPSCs into osteocytes, adipocytes, and chondrocytes (4, 27). The alizarin red staining showed that hDPSCs cultured in both media displayed a substantial mineral deposition after induction, but a significantly higher expression of osteogenic markers (ALP and RUNX) was detected in hDPSCs expanded in AMMS. Consistent with a previous study, hDPSCs cultured in SCM showed a limited adipogenic differentiation potential (4), but hDPSCs expanded in AMMS exhibited more obvious lipid droplet staining and higher marker genes (LPL and PPARγ) expression under adipogenic induction. Chondrogenic differentiation potential of hDPSCs has been extensively explored by using a 3D pellets culture technique (5, 18, 27), and spontaneous chondrogenic differentiation occurred when the pellets were cultured in control medium (5). However, the cartilage spheres formed in the AMMS control were smaller and less condensed, which may be caused by a reduction of mineral deposition and production of collagen in the extracellular matrix (ECM) of the pellets (27). In spite of lower spontaneous chondrogenic potential under chondrogenic induction, both the morphology of the cartilage spheres and the expression of chondrogenic marker genes were comparable between the hDPSCs expanded in SCM and AMMS. Generally, the hDPSCs expanded in AMMS showed a higher osteogenic and adipogenic potential compared to that expanded in SCM.

Due to their neural crest cells origin, hDPSCs are believed to have a high neural cell differentiation potential (13), and they can spontaneously differentiate into neural lineage cells in vitro (7, 24). Consistently, hDPSCs cultured in SCM expressed both neural progenitor markers of Nestin and Vimentin, and mature neuron marker of NeuN. On the other hand, hDPSCs cultured in AMMS exclusively expressed neural progenitor markers (Nestin and Vimentin). This difference may indicate a better stemness maintenance environment in AMMS, and supporting this speculation, aged cells (P20) cultured in AMMS showed a high percentage and intensity of NeuN and GFAP expression (data not shown). The EGF/bFGF/B27 treatment is a simple way to partially induce hDPSCs into mature neural cells (24). But in this study, EGF/bFGF/B27 treatment only enhanced the expression of Nestin and Vimentin. Interestingly, this result is very similar to the hDPSCs cultured in a spheres culture system, which is believed to maintain the cell potency better and to be a good process to produce neural stem cells (28). The very high expression of Nestin and Vimentin of hDPSCs cultured AMMS supplemented with EGF/bFGF/B27 might be an effective method to produce neural stem cell lines with monolayer growing hDPSCs (28), which would contribute to promote the application of hDPSCs derived NSCs in the treatment of neurogenesis diseases (29).

Like other MSCs, hDPSCs can release a plethora of trophic and immunoregulatory factors and it was proposed that their paracrine mediated regenerative effects might be a major mechanism involved in MSCs-based regenerative therapy (30). In this study, we detected the secretory levels of HGF, FGF2, BDNF, GDNF, IL-6, IL-10, PGE2 and TGFβ1, which are representatives of trophic factors, neuroprotective factors and immunoregulatory factors. The results showed a significant difference of HGF, PGE2 and TGFβ1 expression between the hDPSCs cultured in SCM and AMMS. HGF is a pleiotropic cytokine, which can promote cell proliferation, the scattering of cell colonies (25), angiogenesis (31), and act as an immunosuppressive stimulus by negatively affecting dendritic cells (DC) and T lymphocytes (32). The essential mediating role of HGF in MSCs based therapy has also been reported (33, 34). This dual role of cell proliferation and immunosuppression implies that the significantly high level of HGF secretion by the hDPSCs cultured in AMMS may be an indication of more effective cell therapy. MSC-derived PGE2 has been demonstrated to have an inhibitory effect on the early maturation of dendritic cells, suppressive effect on the proliferation of activated lymphocytes, and stimulatory effect on the differentiation of macrophages into M2 phenotype (35). However, the synthesis of PGE2 is induced by an inflammatory environment in vivo (36) or the pro-inflammatory factor—IFNγ (37). Hence, the lower level of PGE2 secreted by hDPSCs cultured in vitro may not reflect a weaker effect on the inflammation regulation, instead that might be caused by the lack of IFNγ stimulation. Transforming growth factor beta 1 (TGFβ1) is constitutively secreted by MSCs, and is also a pleiotropic cytokine. TGFβ1 is involved with regulating the self-renewal properties of stem cells and accelerates the proliferation of mammalian DPSCs in vitro (38). However, there are studies suggesting that TGFβ1 produced by human or rat DPSCs may prevent the self-renewal of DPSCs spheres cultured in a xenogeneic serum-free medium (39, 40). In addition, TGFβ1 expression by hMSCs and the immunosuppressive activity require a direct contact with the T-cell or the trigger by Th1 cytokine interferon (IFN) γ (37). The level of TGFβ1 secreted by hDPSCs cultured in vitro without stimulus might not be a good indicator of either cell proliferation or immunosuppressive activity.

Conclusion

The hDPSCs are considered to be a good cell source for cell-based therapy, due to the advantages of cell properties and easy access. However, a standard good manufacturing practice (GMP) compliant culture system is urgently required for large-scale cell production. In this study, the comprehensive comparison of cell properties of hDPSCs in this study provides strong evidence that the hDPSCs isolated and expanded in a commercially available GMP compliant and xenogeneic serum-free medium (AMMS) possess the characteristics of higher proliferation capacity, differentiation potential and more effective cytokines secretion. This positive result of expanding hDPSCs in vitro, would suggest an excellent culture system, which certainly would promote the application of hDPSCs in the clinical therapy.

Acknowledgements

This research was supported from Key R&D Plan Projects in Zhejiang Province (Development of high-efficiency guided/induced bone repair particles, 2021C04013).

Footnotes

  • Authors’ Contributions

    All the Authors contributed to the study’s conception and design. Data collection, the first draft of the manuscript writing, and analyses of the trilineage differentiation assay/neural cell oriented differentiation assay/enzyme-linked immunosorbent assay were performed by Juan Li. Material preparation, analyses of isolation and expansion of hDPSCs were performed by Xuewei Lv. Analyses of the real-time polymerase chain reaction/SA-β-gal assay were performed by Tingting Ge, and analyses of the CFU assay/immunophenotypic analysis by Jiaman Shi. The formulation or evolution of overarching research goals and aims, supervision of experimental progress, and the revision of the manuscript were performed by Haiyan Lin, Gideon Verwoerd and Yuansong Yu.

  • Conflicts of Interest

    The Authors have no relevant financial or non-financial interests to disclose.

    There is no relevant financial or non-financial conflict of interests related to T&L biotechnology.

  • Received June 28, 2023.
  • Revision received July 29, 2023.
  • Accepted September 1, 2023.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Improved Cell Properties of Human Dental Pulp Stem Cells (hDPSCs) Isolated and Expanded in a GMP Compliant and Xenogeneic Serum-free Medium
JUAN LI, XUEWEI LV, TINGTING GE, JIAMAN SHI, GIDEON VERWOERD, HAIYAN LIN, YUANSONG YU
In Vivo Nov 2023, 37 (6) 2564-2576; DOI: 10.21873/invivo.13364
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